Detection of mage-a expression

ABSTRACT

An oligonucleotide, primer or probe comprises the nucleotide sequences of any of SEQ ID NO. 5, 6, 7, 2, 3, 4, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 25. The oligonucleotides are useful for the detection of the methylation status of a gene, in particular the MAGE-A 3  gene. The oligonucleotides are useful in primer pairs, kits and methods for determining the methylation status of the MAGE-A 3  gene and for diagnosing cancer, directing therapy and selecting subjects for treatment. The primer or probe can comprise a loop or hairpin structure and can be used in real-time methylation specific PCR.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/678,702, filed Jul. 16, 2010, which application was published on Nov.4, 2010, as U.S. Publication No. 20100280105, and which application isthe U.S. national stage application of International ApplicationPCT/GB2908/003142, filed Sep. 17, 2008, which international applicationwas published on Mar. 26, 2009, as International Publication WO2009/037438 in the English language. The International Applicationclaims priority of U.S. Provisional Patent Application No. 60/960,128,filed Sep. 17, 2007, the contents of which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The present invention is concerned with the detection of MAGE-A familygene expression. More specifically, the invention relates to methods ofdetecting methylated or unmethylated forms of MAGE-A3 and associatedoligonucleotides, primers, probes, primer pairs and kits. The methods ofthe invention involve amplification techniques, in particularfluorescence based real-time and end-point pCR methods, and havediagnostic, prognostic and therapeutic utility.

BACKGROUND TO THE INVENTION

MAGE genes belong to the family of cancer/testis antigens. The MAGEfamily of genes comprises over 20 members and is made up of MAGE A, B, Cand D genes (Scanlan et al, (2002) Immunol Rev. 188:22-32; Chomez et al,(2001) Cancer Res. 61 (14):5544-51). They are clustered on chromosome X(Lucas et al., 1998 Cancer Res. 58. 743-752; Lucas et al., (1999 CancerRes 59:4100-4103; Lucas et al., 2000 Int J Cancer 87:55-60; Lurquin etal., 1997 Genomics 46:397-408; Muscatelli et al., 1995 Proc Natl AcadSci USA 92:4987-4991; PoId et al., 1999 Genomics 59:161-167; Rogner etal 1995 Genomics 29:725-731), and have a yet undefined function (Ohmanet al 2001 Exp Cell Res. 265(21: 185-94). The MAGE genes are highlyhomologous and the members of the MAGE-A family, especially, havebetween 60-98% homology.

The human MAGE-A3 gene is expressed in various types of tumours,including melanoma (Furuta et al. 2004 Cancer Sci. 95, 962-968.),bladder cancer, hepatocellular carcinoma (Qiu et al. 2006. ClinicalBiochemistry 39, 259-266), gastric carcinoma (Honda et al. 2004 BritishJournal of Cancer 90, 838-843), colorectal cancer (Kim et al. 2006 WorldJournal of Gastroenterology 12, 5651-5657) and lung cancer (NSCLC)(Scanlan et al 2002 Immunol Rev. 188:22-32; Jang et al 2001 Cancer Res.61, 21: 7959-7963). No expression has been observed in any normal adulttissues with the only exception of testicular germ cells or placenta(Haas et al. 1988 Am J Reprod Immunol Microbiol 18:47-51; Takahashi etal, 1995 Cancer Res 55:3478-382).

Antigen-Specific Cancer Immunotherapeutics (ASCI) represent a novelclass of medicines designed to train the immune system to recognize andeliminate cancer cells in a highly specific manner. As such, ASCI allowtargeted treatment. ASCI have two principal components: “tumor antigens”to direct the immune response specifically against the cancer cell and“adjuvant systems” that comprise immuno-stimulation compounds selectedto increase the anti-tumour immune response. MAGE-A3 antigen andconstructs suitable for use in ASCI are described in WO99/40188 andencouraging phase II study results with MAGE-A3 ASCI in patients withNon Small Lung Cancer (NSCLC) have been reported recently (J. Clin.Oncol. Vol. 25, No. 18S (June 20 Suppl.) 2007: 7554).

It is important to have quantitative high throughput assays capable ofspecifically identifying MAGE-A3-expressing patients that would benefitfrom immunotherapy, monitoring MAGE-A3 expression for dosage purpose,identifying Mage-A3 expressing samples in clinical trials, or simplyidentify at an early stage patients with cancer. A number of applicablediagnostic methods have been described and include: Semi-quantitativeRT-PCR (De Plaen et al. 1994 Immunogenetics 40(5);360-9), other PCRbased techniques and low-density microarrays (Zammatteo et al. 2002Clinical Chemistry 48(1) 25-34). Further, an improved RT-PCR method foruse in conjunction with MAGE-A3 ASCI has been discussed inWO2007/147876.

The greatest disadvantage of the existing assays is that they requireRNA isolation to assess MAGEA3 expression. Formalin-Fixed,Paraffin-Embedded (FFPE) tumour tissue is the usual method of tumourtissue preservation within clinical centres. The fixation in formalinchanges the structure of molecules of RNA within the tissue, causingcross linking and also partial degradation. The partial degradationleads to the creation of smaller pieces of RNA of between 100-300 basepairs. These structural changes to the RNA limit the use of RNAextracted from FFPE tissue to measure MAGEA3 expression levels.

An object of the present invention is to provide an improved assay thateliminates the disadvantages of the existing assays.

Gene methylation is an important regulator of gene expression. Inparticular, methylation at cytosine residues found in CpG di-nucleotidepairs in the promoter region of specific genes can contribute to manydisease conditions through down regulation of gene expression. Foxexample, aberrant methylation of tumour suppressor genes can lead to upor down regulation of these genes and is thus associated with thepresence and development of many cancers (Hoffmann et al. 2005 BiochemCell Biol 83: 296-321). Patterns of aberrant gene methylation are oftenspecific to the tissue of origin. Accordingly, detection of themethylation status of specific genes may be of prognostic and diagnosticutility and can be used to both determine the relative stage of adisease and also to predict response to certain types of therapy (Laird,2003 Nat Rev Cancer 3: 253-266).

Methylation-Specific PGR (MSP) with visualization of the results on agel (gel-based MSP assay) is widely used to determine epigeneticsilencing of genes (Esteller M et al. Cancer Res 2001;61:3225-9.),although quantitative tests using other technologies have been developed(Laird P W., Nat Rev Cancer 2003; 3:253-66; Eads et al. Nucleic AcidsRes 2000;28:E32; Mikeska T. et al. J Mol Diagn 2007).

A number of fluorescence based technologies are available for real-timemonitoring of nucleic acid amplification reactions. One such technologyis described in U.S. Pat. No. 6,090,552 and EP 0912597 and iscommercially known as Amplifluor®. This method is also suitable forend-point monitoring of nucleic acid amplification reactions.Vlassenbroeck et al. (Vlassenbroeck et al., 2008. Journal of Molec.Diagn., V10, No. 4) further describes a standardized direct, real-timeMSP assay with use of the Amplifluor® technology.

SUMMARY OF THE INVENTION

The present invention relates to improved methods and/or assays ofmeasuring MAGE-A3 expression. The present invention further relates tocertain types of therapy, in particular Antigen-Specific CancerImmunotherapeutics (ASCI) based treatment of patients identified asexpressing MAGE-A3 through use of oligonucleotides, primers, probes,primer pairs, kits and/or methods as described herein. MAGE-A3 proteinexpression is detected by determining the methylation status of theMAGE-A3 gene rather than measuring the expression level of the geneitself. The inventors show that the methylation status result of theirmethylation tests is in good concordance with results obtained with theRT-PCR assay that established the predictive value of the MAGE-A3expression in NSCLC for benefit from MAGE-A3 immunotherapy. The assaysare thus useful for selecting patients suitable for treatment, forpredicting the likelihood of successful treatment of a patient and canbe used to aid patient therapy selection.

In one aspect, the present invention provides an oligonucleotide, primeror probe comprising or consisting essentially of or consisting of thenucleotide sequence of any SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12,13, 14, 15, 16, 17, 18, 19 or 25 which oligonucleotide, primer or probeis useful for the detection of the methylation status of a gene.

The oligonucleotide, primer or probe preferably comprises, consistsessentially of or consists of the following contiguous sequences in 5′to 3′ order:

-   -   (a) a first nucleotide sequence of between approximately 6 and        30 nucleotides, wherein a nucleotide within said first        nucleotide sequence is labelled with a first moiety selected        from the donor moiety and the acceptor moiety of a molecular        energy transfer pair, wherein the donor moiety emits        fluorescence at one or more particular wavelengths when excited,        and the acceptor moiety absorbs and/or quenches said        fluorescence emitted by said donor moiety;    -   (b) a second, single-stranded nucleotide sequence comprising,        consisting essentially of or consisting of between approximately        3 and 20 nucleotides;    -   (c) a third nucleotide sequence comprising, consisting        essentially of or consisting of between approximately 6 and 30        nucleotides, wherein a nucleotide within said third nucleotide        sequence is labelled with a second moiety selected from said        donor moiety and said acceptor moiety, and said second moiety is        the member of said group not labelling said first nucleotide        sequence, wherein said third nucleotide sequence is        complementary in reverse order to said first nucleotide sequence        such that a duplex can form between said first nucleotide        sequence and said third nucleotide sequence such that said first        moiety and second moiety are in proximity such that, when the        donor moiety is excited and emits fluorescence, the acceptor        moiety absorbs and quenches said fluorescence emitted by said        donor moiety; and

(d) at the 3′ end of the primer, a fourth, single-stranded nucleotidesequence comprising, consisting essentially of or consisting of betweenapproximately 8 and 40 nucleotides that comprises at its 3′ end thenucleotide sequence of any of SEQ ID NO. 2, 4, 5, 7, 8, 11, 13, 14, 16,17, 19 or 25;

-   -   wherein when said duplex is not formed, said first moiety and        said second moiety are separated by a distance that prevents        molecular energy transfer between said first and second moiety.

The specific nucleotide sequences at the 3′ end permit the methylationstatus of the MAGE-A3 gene to be determined. These primers bindpreferentially to unmethylated forms of the MAGEA3 gene followingtreatment with an appropriate reagent (as discussed herein). Propertiesof these oligonucleotides are discussed herein, which discussion appliesmutatis mutandis. The specific nucleotide sequences are able to primesynthesis by a nucleic acid polymerase of a nucleotide sequencecomplementary to a nucleic acid strand comprising the portion of themethylated or unmethylated DNA of the MAGE A family gene.

Most preferably, the oligonucleotide, primer or probe consists of thenucleotide sequence of SEQ ID NO. 3, 6, 9, 12, 15 or 18 and is used toamplify a portion of the gene of interest.

Further provided is a primer pair comprising a primer comprising orconsisting essentially of or consisting of the nucleotide sequence ofany SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18,19 or 25.

In a further aspect, there is provided a kit comprising at least oneprimer, primer pair or set of primers comprising or consistingessentially of or consisting of the nucleotide sequence of any SEQ IDNO. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 25.The kit is for detecting the methylation status of a gene, in particulara MAGE-A family gene such as MAGE-A3.

In a further aspect, the invention provides for a method of detectingthe methylation status of the Mage-A3 gene in a DNA-containing sample,comprising:

-   -   (a) contacting/treating the DNA-containing sample with a reagent        which selectively modifies unmethylated cytosine residues in the        DNA to produce detectable modified residues but which does not        modify methylated cytosine residues    -   (b) amplifying at least a portion of the methylated or        unmethylated gene of interest using at least one primer pair, at        least one primer of which is designed to bind only to the        sequence of methylated or unmethylated DNA respectively        following treatment with the reagent, wherein at least one        primer in the primer pair comprises, consists essentially of, or        consists of a the nucleotide sequence of any of SEQ ID NO. 2, 3,        4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 25 (as        appropriate).

In a further aspect, there is provided a method of diagnosing cancer orpredisposition to cancer comprising detecting the methylation status ofthe MAGE-A3 gene in a sample by using an oligonucleotide, primer orprobe, primer pair, kit or a method as described herein, wherein thepresence or unmethylated MAGE-A3 in the sample is indicative for canceror predisposition to cancer.

In a further aspect, there is provided a method for determining thepresence of MAGE-A3 positive tumor comprising detecting the methylationstatus of the MAGE-A3 gene in a sample by using an oligonucleotide,primer or probe, primer pair, kit or a method as described herein,wherein the presence of unmethylated MAGE-A3 is indicative for thepresence of a MAGE-A3 positive tumor. By “MAGE-A3 positive tumor” ismeant any tumor or tumor cells (isolated from a patient) which expressthe MAGE-A3 antigen.

The invention further provides a method for identifying and/or selectinga patient suitable for treatment with a MAGE-A3 immunotherapeuticcomprising detecting the methylation status of the MAGE-A3 gene in asample of the patient by using an oligonucleotide, primer or probe,primer pair, kit or a method as described herein, wherein if the MAGE-A3gene is unmethylated the subject is (preferably) identified and/orselected for treatment with the MAGE-A3 immunotherapeutic. Thus,patients with unmethylated MAGEA3 are preferred to those in which thegene is methylated.

Alternatively, if the gene is not unmethylated the subject is preferablynot identified and/or selected for treatment with a MAGE-A3immunotherapeutic.

In a related aspect, the invention provides a method for predicting thelikelihood of successful treatment of cancer comprising detecting themethylation status of the MAGE-A3 gene in a sample of the patient byusing an oligonucleotide, primer or probe, primer pair, kit or a methodas described herein, wherein if the gene is unmethylated the likelihoodof successful treatment with a MAGE-A3 immunotherapeutic is higher thanif the gene is methylated.

Alternatively, the absence of unmethylated MAGE-A3 in the sampleindicates that the likelihood of resistance to treatment with a MAGE-A3immunotherapeutic is higher than if the gene is unmethylated. Thus, thedetection of a methylated MAGE-A3 gene indicates that the probability ofsuccessful treatment with an immunotherapeutic is low.

In a further related aspect, the invention provides a method ofselecting a suitable treatment regimen for cancer comprising detectingthe methylation status of the MAGE-A3 gene in a sample of the patient byusing an oligonucleotide, primer or probe, primer pair, kit or a methodas described herein, wherein if the gene is unmethylated, animmunotherapeutic is selected for treatment.

Alternatively, if the gene is not unmethylated, treatment with animmunotherapeutic is contra-indicated.

Also provided is a method of treating cancer in a subject comprisingadministration of an immunotherapeutic, wherein the subject has beenselected for treatment on the basis of measuring the methylation statusof a MAGE-A3 gene, according to any of the methods of the invention orby using an oligonucleotide, primer or probe, primer pair, kit or amethod as described herein.

Preferably, for all of the different aspects described herein, thedetection of unmethylated MAGE-A3 gene corresponds to an increased levelof MAGE-A3 protein.

The present invention further provides a method of treating a patientcomprising: measuring the methylation status of a MAGE-A3 gene accordingto any of the methods of the invention by using an oligonucleotide,primer or probe, primer pair, kit or a method as described herein, andthen administering to the patient a composition comprising MAGE-A3 asdescribed herein. The composition is preferably administered if theMAGE-A3 gene is found to be unmethylated.

In a further aspect there is provided a method of treating a patientsusceptible to recurrence of a MAGE-A3 expressing tumour, the patienthaving been treated to remove tumour tissue, the method comprising:measuring the methylation status of a MAGE-A3 gene in the tumour tissue,according to any of the methods of the invention or by using anoligonucleotide, primer or probe, primer pair, kit or a method asdescribed herein, and then administering to the patient a compositioncomprising MAGE-A3 as described herein. The composition is preferablyadministered if the MAGE-A3 gene is found to be unmethylated.

In a still further aspect of the present invention there is provided ause of a composition comprising MAGE-A3 in the manufacture of amedicament for the treatment of a patient suffering from a tumour, inwhich a patient has been selected for treatment on the basis ofmeasuring the methylation status of a MAGE-A3 gene, according to any ofthe methods of the invention or by using an oligonucleotide, primer orprobe, primer pair, kit or a method as described herein. There is alsoprovided a composition comprising MAGE-A3 for use in the treatment of apatient suffering from a tumour, in which a patient has been selectedfor treatment on the basis of measuring the methylation status of aMAGE-A3 gene, according to any of the methods of the invention or byusing an oligonucleotide, primer or probe, primer pair, kit or a methodas described herein.

In a yet further embodiment there is provided a use of a compositioncomprising MAGE-A3 in the manufacture of a medicament for the treatmentof a patient susceptible to recurrence of a MAGE-A3 expressing tumour,in which a patient has been selected for treatment on the basis ofmeasuring the methylation status of a MAGE-A3 gene, according to any ofthe methods of the invention or by using an oligonucleotide, primer orprobe, primer pair, kit or a method as described herein. There is alsoprovided a composition comprising MAGE-A3 for use in the treatment of apatient susceptible to recurrence of a MAGE-A3 expressing tumour, inwhich a patient has been selected for treatment on the basis ofmeasuring the methylation status of a MAGE-A3 gene, according to any ofthe methods of the invention or by using an oligonucleotide, primer orprobe, primer pair, kit or a method as described herein.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an assay for detecting the presence and/or amountof a methylated or unmethylated MAGE-A3 gene in a DNA-containing sample.To develop this assay, it was necessary to identify regions susceptibleto methylation in the MAGE-A3 gene and to develop particularoligonucleotides that could differentiate unmethylated from methylatedforms of the MAGE-A3 gene.

Accordingly, in a first aspect, the invention provides oligonucleotidescomprising, consisting essentially of, or consisting of the nucleotidesequence of any SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15,16, 17, 18, 19 or 25. These oligonucleotides are useful in the detectionof the methylation status of a gene of interest. The oligonucleotidesmay serve as primers and/or probes. In certain embodiments, theoligonucleotides detect the unmethylated form to the gene. Suitableoligonucleotides to detect the unmethylated form comprise, consistessentially of, or consist of the nucleotide sequence of any of SEQ IDNO. 2, 4, 5, 7, 8, 11, 13 or 25. Suitable oligonucleotides to detect themethylated form comprise, consist essentially of, or consist of thenucleotide sequence of any SEQ ID NO. 14, 16, 17 or 19. In certainembodiments, these oligonucleotides comprise a hairpin structure asdescribed in the present invention. Such preferred oligonucleotidesinclude the sequences according to SEQ ID NO. 3, 6, 9, and 12 fordetecting the unmethylated form of the gene and SEQ ID NO. 15 and 18 fordetecting the methylated form of the gene.

The “genes” or “gene of interest” of the invention are preferably MAGEA3and/or MAGEA6 genes. MAGEA3 and MAGEA6 are the gene symbols approved bythe HUGO Gene Nomenclature Committee. The MAGEA3 gene is located onchromosome X (location g28) and the gene sequence is listed under theaccession numbers NM_(—)005362 and ENSG00000197172. The MAGE-A3 geneencodes melanoma antigen family A, 3. The MAGEA6 gene is located onchromosome X (location q28). MAGE-A3 is often referred tointerchangeably as MAGE-3 or MAGEA3. Likewise, MAGE-A6 is often referredto interchangeably as MAGE-6 or MAGEA6; all are used herein.Hypomethylation of these genes may be linked to the incidence ofcancers, such as melanoma or lung cancer (including NSCLC) for example.

CpG dinucleotides susceptible to methylation are typically concentratedin the promoter region of human genes. In a preferred embodiment, themethylation status of the gene is assessed by determining levels ofmethylation in the promoter region of the gene. A “promoter” is a regionupstream from the transcription start site (TSS), extending betweenapproximately 10 Kb, 3Kb, 1 Kb, 500 bp or 150 to 300 bp from the TSS.For MAGE-3, the CpG distribution in the promoter region is ratherscarce.

The term “methylation state” or “methylation status” refers to thepresence or absence of 5-methylcytosine (“5-mCyt”) at one or a pluralityof CpG dinucleotides within a DNA sequence. “Hypermethylation” isdefined as an increase in the level of methylation above normal levels.Thus, it relates to aberrant methylation of cytosine (5-mCyt) atspecific CpG sites in a gene, often in the promoter region. Normallevels of methylation may be defined by determining the level ofmethylation in non-cancerous cells for example.

“Hypomethylation” refers to a decreased presence of 5-mCyt at one or aplurality of CpG dinucleotides within a DNA sequence (of a test DNAsample), relative to the amount of 5-mCyt found at corresponding CpGdinucleotides within a “normal” DNA sequence (found in a suitablecontrol sample). Again, “normal” levels of methylation may be defined bydetermining the level of methylation in non-cancerous cells for example.In this invention, hypomethylation of the MAGE-A3and/or MAGE-A6 gene isindicative of an increased expression of this tumour associated antigengene which provides a reliable indicator of cancer.

The invention provides in a second aspect for a method of detecting thepresence and/or amount of a methylated or unmethylated gene in aDNA-containing sample comprising the step of contacting theDNA-containing sample with at least one oligonucleotide comprising,consisting essentially of, or consisting of the nucleotide sequence ofany SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18,19 or 25. The method preferably comprises the further step of assessingwhether the gene is methylated or unmethylated. This may depend uponwhether the oligonuclotide stably binds to the DNA in the DNA-containingsample, as discussed herein.

Techniques tor assessing methylation status are based on distinctapproaches. Any suitable technique, applying the oligonucleotides of theinvention may be employed. In one embodiment, approaches for detectingmethylated CpG dinucleotide motifs use a reagent which selectivelymodifies unmethylated cytosine residues in the DNA to produce detectablemodified residues. The reagent does not modify methylated cytosineresidues and thus allows for discrimination between unmethylated andmethylated nucleic acid molecules in a downstream process, whichpreferably may involve nucleic acid amplification. The reagent may, inone embodiment, act to selectively deaminate unmethylated cytosineresidues. Thus, following exposure to the reagent the unmethylated DNAcontains a different nucleotide sequence to that of correspondingmethylated DNA. The deamination of cytosine results in a uracil residuebeing present, which has the same base pairing properties as thymine,which differs from cytosine base pairing behaviour. This makes thediscrimination between methylated and non-methylated cytokines possible.

Useful conventional techniques for assessing sequence differences useoligonucleotide primers. Two approaches to primer design are possible.Firstly, primers may be designed that themselves do not cover anypotential sites of DNA methylation. Sequence variations at sites ofdifferential methylation are located between the two primer bindingsites and visualisation of the sequence variation requires further assaysteps. Secondly, primers may be designed that hybridize specificallywith either the methylated or unmethylated version of the initialtreated sequence. After hybridization, an amplification reaction can beperformed and amplification products assayed using any detection systemknown in the art. The presence of an amplification product indicatesthat the primer hybridized to the DNA. The specificity of the primerindicates whether the DNA had been modified or not, which in turnindicates whether the DNA had been methylated or not. If there is asufficient region of complementarity, e.g., 12, 13, 14, 15, 16, 17, 18,19, 20, 21 or 22 nucleotides, to the target, then the primer may alsocontain additional nucleotide residues that do not interfere withhybridization but may be useful for other manipulations. Examples ofsuch other residues may be sites for restriction endonuclease cleavage,for ligand binding or for factor binding or linkers or repeats, orresidues for purpose of visualisation. The oligonucleotide primers mayor may not be such that they are specific for modified methylatedresidues. Preferred oligonucleotides for use as primers comprise,consist essentially of, or consist of the nucleotide sequence of any ofSEQ. ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19or 25.

A further way to distinguish between modified and unmodified nucleicacid is to use oligonucleotide probes. Such probes may hybridizedirectly to modified nucleic acid or to further products of modifiednucleic acid, such as products obtained by amplification. Probe-basedassays exploit the oligonucleotide hybridisation to specific sequencesand subsequent detection of the hybrid. There may also be furtherpurification steps before the amplification product is detected e.g. aprecipitation step. Oligonucleotide probes may be labeled using anydetection system known in the art. These include but are not limited tofluorescent moieties, radioisotope labelled moieties, bioluminescentmoieties, luminescent moieties, chemiluminescent moieties, enzymes,substrates, receptors, or ligands. Preferred oligonucleotides for use asprobes comprise, consist essentially of, or consist of the nucleotidesequence of any of SEQ ID NO. 2, 4, 5, 6, 7, 8, 11, 13, 14, 16, 17, 19or 25.

“Oligonucleotide primer” is referred to herein interchangeably as“primer”. Likewise, “oligonucleotide probe” is referred to hereininterchangeably as “probe”.

In preferred embodiments, the methylation status of the gene (or portionthereof, especially the CpG islands) is determined using methylationspecific PCR (MSP).

The MSP technique will be familiar to one of skill in the art. In theMSP approach, DNA may be amplified using primer pairs designed todistinguish unmethylated from methylated DNA by taking advantage ofsequence differences as a result of sodium-bisulfite treatment (Herman JG et al. Proc Natl Acad Sci U S A. 1996 Sep. 3; 93 (18):9821-6 and WO97/46705). A specific example of the MSP technique is designatedreal-time quantitative MSP (QMSP), which permits reliable quantificationof methylated DNA in real time.

Real-time methods are generally based on the continuous opticalmonitoring of an amplification procedure and utilise fluorescentlylabelled reagents whose incorporation in a product can be quantified andwhose quantification is indicative of copy number of that sequence inthe template. Such labeled reagent may be a fluorescent dye thatpreferentially binds double-stranded DNA and whose fluorescence isgreatly enhanced by binding of double-stranded DNA. Alternatively,labeled primers and/or labeled probes can be used. They represent aspecific application of the wail known and commercially availablereal-time amplification techniques such as TAQMAN®, MOLECULAR BEACONS®,AMPLIFLUOR® and SCORPION® DzyNA®, etc. Often, these real-time methodsare used with the polymerase chain reaction (PCR).

TaqMan technology uses linear, hydrolytic oligonucleotide probes thatcontain a fluorescent dye and a quenching dye. When irradiated, theexcited fluorescent dye transfers energy to the nearby quenching dyemolecule rather than fluoresencing (FRET principle). TaqMan probesanneal to an internal region of the PCR product and are cleaved by theexonuclease activity of the polymerase when it replicates a template.This ends the activity of the quencher, and the reporter dye starts toemit fluorescence which increases in each cycle proportional to the rateof probe cleavage.

Molecular beacons also contain fluorescent and quenching dyes, but theyare designed to adopt a hairpin structure while tree in solution tobring both dyes in close proximity for Fluorescence Resonance EnergyTransfer (FRET) to occur. When the beacon hybridises to the targetduring the annealing step, the hairpin linearises and both dyes (donorand acceptor/quencher) are separated. The increase in fluorescencedetected from the donor will correlate to the amount of PCR productavailable.

With scorpion probes, sequence-specific priming and PCR productdetection is achieved using a single oligonucleotide. The scorpion probemaintains a stem-loop configuration in the unhybridized state and FREToccurs between the fluorophore and quencher. The 3′ portion of the stemalso contains a sequence that is complementary to the extension productof the primer. This sequence is linked to the 5′ end of a specificprimer via a non-amplifiable monomer. After extension of the scorpionprimer, the specific probe sequence is able to bind to its complementWithin the extended amplicon, thus opening up the hairpin loop,separating the fluorophore and quencher, and providing a fluorescencesignal.

In Heavymethyl, the priming is methylation specific, but non-extendableoligonucleotide blockers provide this specificity instead of the primersthemselves. The blockers bind to bisulfite-treated DNA in amethylation-specific manner, and their binding sites overlap the primerbinding sites. When the blocker is bound, the primer cannot bind andtherefore the amplicon is not generated. Heavymethyl can be used incombination with real-time detection.

The Plexor™ qPCR and qRT-PCR Systems take advantage of the specificinteraction between two modified nucleotides to achieve quantitative PCRanalysis. One of the PCR primers contains a fluorescent label adjacentto an iso-dC residue at the 5′ terminus. The second PCR primer isunlabeled. The reaction mix includes deoxynucleotides and iso-dGTPmodified with the quencher dabcyl. Dabcyl-iso-dGTP is preferentiallyincorporated at the position complementary to the iso-dC residue. Theincorporation of the dabcyl-iso-dGTP at this position results inquenching of the fluorescent dye on the complementary strand and areduction in fluorescence, which allows quantitation duringamplification. For these multiplex reactions, a primer pair with adifferent fluorophore is used for each target sequence.

Thus the oligonucleotides comprising, consisting essentially of, orconsisting of the nucleotide sequence of any SEQ ID NO. 2, 3, 4, 5, 6,7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 25 may be employed asprimers or probes in the aforementioned methods for detection of themethylation status of a gene of interest.

In a preferred embodiment, the invention provides a real-time method ofdetecting the presence and/or amount of a methylated or unmethylatedgene of interest in a DNA-containing sample, comprising:

-   -   (a) contacting/treating the DNA-containing sample with a reagent        which selectively modifies unmethylated cytosine residues in the        DNA to produce detectable modified residues but which does not        modify methylated cytosine residues    -   (b) amplifying at least a portion of the methylated or        unmethylated gene of interest using at least one primer pair, at        least one primer of which is designed to bind only to the        sequence of methylated or unmethylated DNA respectively        following treatment with the reagent, wherein at least one        primer in the primer pair comprises, consists essentially of, or        consists of the nucleotide sequence of any of SEQ ID NO. 2, 3,        4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18 or 19.

The gene of interest in the methods of the invention is preferably theMAGE-A3 and/or the MAGEA-6 gene. Preferably, at least one primer in theprimer pair is a primer containing a stem loop structure carrying adonor and an acceptor moiety of a molecular energy transfer pairarranged such that in the absence of amplification, the acceptor moietyquenches fluorescence emitted by the donor moiety (upon excitation) andduring amplification, the stem loop structure is disrupted so as toseparate the donor and acceptor moieties sufficiently to produce adetectable fluorescence signal. This may be detected in real-time toprovide an indication of the presence of the methylated or unmethylatedgene of interest. The primer in the primer pair which comprises,consists essentially of, or consists of the nucleotide sequence of anyof SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18 or19 preferably carries the stem loop structure.

In certain embodiments the gene copy number of the methylated orunmethylated gene is determined. Here, the method described hereinpreferably comprises a further step: (c) quantifying the results of thereal-time detection against a standard curve for the methylated orunmethylated gene of interest to produce an output of gene copy number.

Preferably, step (c) is further characterised in that the amplificationis considered valid where the cycle threshold value is less than 40.

For genes such as the MageA3 and/or MageA6 gene, detection of anunmethylated version of the gene may be of primary relevance.

The methods of the invention allow the presence of a methylated orunmethylated gene of interest to be detected in a sample in real-time.Since the methods of the invention are quantitative methods, the(relative) amounts of the methylated or unmethylated form of the gene ofinterest can also be determined as the reaction proceeds. Real-timemethods do not need to be utilised, however. Analyses can be performedonly to discover whether the target DNA is present in the sample or not.End-point amplification detection techniques utilise the same approachesas widely used for Real Time PCR. Therefore, the methods of theinvention may encompass an end-point method of detecting the presenceand/or amount of a methylated or unmethylated gene of interest in aDNA-containing sample.

Thus, the invention provides a(n end point) method of detecting thepresence and/or amount of a methylated or unmethylated gene of interestin a DNA-containing sample, comprising:

-   -   (a) contacting and/or treating the DNA-containing sample with a        reagent which selectively modifies unmethylated cytosine        residues in the DNA to produce detectable modified residues but        which does not modify methylated cytosine residues    -   (b) amplifying at least a portion of the methylated or        unmethylated gene of interest using at least one primer pair, at        least one primer of which is designed to bind only to the        sequence of methylated or unmethylated DNA respectively        following treatment with the reagent, wherein at least one        primer in the primer pair comprises, consists essentially of, or        consists of the nucleotide sequence of any of SEQ ID NO. 2, 3,        4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 25.

As aforementioned, the gene of interest in the methods of the inventionis preferably the MAGE-A3 and/or MAGE-A6 gene. Preferably, at least oneprimer in the primer pair is a primer containing a stem loop structurecarrying a donor and an acceptor moiety of a molecular energy transferpair having the characteristics as described herein. The primer in theprimer pair which comprises, consists essentially of, or consists of thenucleotide sequence of any of SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12,13, 14, 15, 16, 17, 18 or 19 preferably carries the stem loop structure.

For the MAGE-A3 and/or MAGE-A6 gene, detection of an unmethylatedversion of the gene may be of primary relevance. Primers comprising,consisting essentially of, or consisting of the nucleotide sequence ofany of SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 25 have beendesigned for the purpose of detecting unmethylated MAGEA3 DNA followingtreatment with the reagent.

The absence of unmethylated gene will indicate the presence ofmethylated gene. However, the detection of methylated gene is alsowithin the scope of the invention. Primers comprising, consistingessentially of, or consisting of the nucleotide sequence of any SEQ IDNO. 14, 15, 16, 17, 18 or 19 have been designed for the purpose ofdetecting methylated MAGEA3 DNA following treatment with the reagent. Incase a gene copy number of the methylated or unmethylated gene isdesired, the method preferably comprises a further step:

-   -   (c) quantifying the results of the detection against a standard        curve for the methylated or unmethylated gene of interest to        produce an output of gene copy number.

All embodiments of the invention are applicable to the end-point aspectsof the invention and thus apply mutatis mutandis. End point analysis mayinvoke use of a fluorescent plate reader or other suitableinstrumentation to determine the fluorescence at the end of theamplification.

The methods of the invention are most preferably ex vivo or in vitromethods carried out on any suitable (DNA containing) test sample. In oneembodiment, however, the method may also include the step of obtainingthe sample. The test sample is a DNA-containing sample, in particular aDNA-containing sample including the gene of interest. The methods of theinvention can be used in the diagnosis of disease, in particular wheremethylation of a gene of interest is (known to be) linked to theincidence of disease.

The DNA-containing sample may comprise any suitable tissue sample orbody fluid. Preferably, the test sample is obtained from a humansubject. For cancer applications, the sample may comprise a tissuesample taken from the tissue suspected of being cancerous or from arepresentative bodily fluid. Hypomethylation of the MAGE-A3 gene hasbeen associated with lung cancer. Thus, in one embodiment, the testsample to be used in the methods of the invention involving a MAGE-A3gene preferably contains lung cells or nucleic acid from lung cells.Most preferably, the sample is a Formalin Fixed Paraffin Embedded (FFPE)tissue. There are two types of lung cancer: non-small cell lung cancer(NSCLC) and small cell lung cancer (SCLC). The names simply describe thetype of cell found in the tumours. The test sample preferably containscells or nucleic acid from non-small cell lung carcinoma (NSCLC). NSCLCincludes squamous-cell carcinoma, adenocarcinoma, and large-cellcarcinoma and accounts for around 80% of lung cancers. In a preferredembodiment, where the cancer is a NSCLC, the sample is a lung tissuesample or a sputum sample. NSCLC is hard to cure and treatmentsavailable tend to have the aim of prolonging life as far as possible andrelieving symptoms of disease. NSCLC is the most common type of lungcancer and is associated with poor outcomes.

Hypomethylation of the MAGE-A3 gene is also linked to bladder cancer.Thus, in additional embodiments, a further preferred test sample to beused in the methods of the invention contains transitional bladder cellsor squamous carcinoma bladder cells. Preferably, the test sample isobtained from a bladder tissue. More preferably, it is derived fromurine and contains nucleic acid from transitional bladder cells orsquamous carcinoma bladder cells. The test sample can be derived fromliquid urine, a precipitate thereof, or a precipitate in the urine. Thetissues and body fluids can be collected using any suitable method, manyof which are well known in the art.

Hypomethylation of the MAGE-A3 gene is also linked to melanoma. Melanomais a pigmented, readily accessible lesion that has been well defined inhistopathological terms. Early radial growth phase (RGP) melanomas caninvade into the epidermis and papillary dermis, but have no capacity formetastasis; resection at this stage is almost completely curative. Asubsequent vertical growth phase (VGP) denotes a transition to a moreaggressive stage, which is capable of metastasis. Changes in geneexpression occurring at the RGP/VGP transition are, thus, of greatinterest. Thus, in additional embodiments, a further preferred testsample to be used in the methods of the invention, contains melanomacells. Preferably, the test sample is obtained from a skin lesion.

Other DNA-containing samples for use in the methods of the inventioninclude samples for diagnostic, prognostic, or personalised medicinaluses. These samples may be obtained from surgical samples, such asbiopsies or fine needle aspirates, from paraffin embedded tissues, fromfrozen tumor tissue samples, from fresh tumour tissue samples or from afresh or frozen body fluid, for example. Non-limiting examples includewhole blood, bone marrow, cerebrospinal fluid, peritoneal fluid, pleuralfluid, lymph fluid, serum, plasma, urine, chyle, stool, ejaculate,sputum, nipple aspirate, saliva, swabs specimens, colon wash specimensand brush specimens. The tissues and body fluids can be collected usingany suitable method, many such methods are well known in the art.Assessment of a paraffin-embedded specimen can be performed directly oron a tissue section. The terms “sample”, “patient sample” and “sample ofthe patient” are used interchangeably and are intended to mean aDNA-containing sample from a patient, as described above.

The methods of the invention may be carried out on purified orunpurified DNA-containing samples. However, in a preferred embodiment,prior to step (a) (the reagent treatment step) or as a preliminary step,DNA is isolated/extracted/purified from the DNA-containing sample. Anysuitable DNA isolation technique may be utilised. Examples ofpurification techniques may be found in standard texts such as MolecularCloning—A Laboratory Manual (Third Edition), Sambrook and Russell (seein particular Appendix 8 and Chapter 5 therein). In one preferredembodiment, purification involves alcohol precipitation of DNA.Preferred alcohols include ethanol and isopropanol. Suitablepurification techniques also include salt-based precipitation methods.Thus, in one specific embodiment the DNA purification techniquecomprises use of a high concentration of salt to precipitatecontaminants. The salt may comprise, consist essentially of or consistof potassium acetate and/or ammonium acetate for example. The method mayfurther include steps of removal of contaminants which have beenprecipitated, followed by recovery of DNA through alcohol precipitation.

In an alternative embodiment, the DNA purification technique is basedupon use of organic solvents to extract contaminants from cell lysates.Thus, in one embodiment, the method comprises use of phenol, chloroformand isoamyl alcohol to extract the DNA. Suitable conditions are employedto ensure that the contaminants are separated into the organic phase andthat DNA remains in the aqueous phase. Further kits use magnetic beads,silica-membrane, etc. Such kits are well known in the art andcommercially available. The methods of the invention may use thePUREGENE® DNA Purification Kit.

In preferred embodiments of these purification techniques, extracted DNAis recovered through alcohol precipitation, such as ethanol orisopropanol precipitation.

Formalin-Fixed, Paraffin-Embedded (FFPE) tumour tissue is the usualmethod of tumour tissue preservation within clinical centres. Such FFPEembedded samples require a dewaxing step prior to DNA extraction. In apreferred embodiment, FFPE tissue samples or sample material immobilizedon slides are first dewaxed by xylene treatment. The contact period withthe xylene should be sufficient to allow the xylene to contact andinteract with the sample. In a more preferred embodiment, FFPE samplesare deparaffinized in 100% xylene for about 2 hours. This step may berepeated once more to ensure complete deparaffinization. After xylenetreatment samples are rehydrated using 70% ethanol.

The methods of the invention may also, as appropriate, incorporate (alsoprior to step (a) or as a preliminary step) quantification ofisolated/extracted/purified DNA in the sample. Quantification of the DNAin the sample may be achieved using any suitable means. Quantitation ofnucleic acids may, for example, be based upon use of aspectrophotometer, a fluorometer or a UV transilluminator. Examples ofsuitable techniques are described in standard texts such as MolecularCloning—A Laboratory Manual (Third Edition), Sambrook and Russell (seein particular Appendix 8 therein). In a preferred embodiment, kits suchas the Picogreen® dsDNA quantitation kit available from MolecularProbes, Invitrogen may be employed to quantify the DNA.

The methods of the invention rely upon a reagent which selectivelymodifies unmethylated cytosine residues in the DNA to produce detectablemodified residues. The mode of action of the reagent has been explainedalready. In a preferred embodiment, the reagent which selectivelymodifies unmethylated cytosine residues in the DNA to produce detectablemodified residues but which does not modify methylated cytosine residuescomprises, consists essentially of or consists of a bisulphite reagent(Frommer et al., Proc. Natl. Acad. Sci. USA 1992 89:1827-1831,). Severalbisulphite containing reagents are known in the art and suitable kitsfor carrying out the deamination reaction are commercially available(such as the EZ DNA methylation kit from Zymo Research). A particularlypreferred reagent for use in the methods of the invention comprises,consists essentially of or consists of sodium bisulphite.

Once the DNA in the sample has been treated with the reagent, it is thennecessary to detect the difference in nucleotide sequence caused by thereagent. This is done using a nucleic acid amplification technique. Asmentioned already, functionally relevant methylation is most commonlyassociated with the promoter regions of genes. In particular, so called“CpG islands” include a relatively high incidence of CpG residues andare often found in the promoter region of the gene. Various softwareprograms exist to allow CpG islands in a gene of interest to beidentified. Accordingly, the methods of the invention may involveamplifying at least a portion of the methylated or unmethylated gene ofinterest using at least one primer pair. As discussed above, since theresidues of interest whose methylation status is to be investigated, aretypically found in defined CpG islands and/or in the promoter region ofthe gene of interest, the primer pair will typically amplify only aportion of the gene (in this region), rather than the entirety. Anysuitable portion of the gene may be amplified according to the methodsof the invention, provided that the amplification product is detectableas a reliable indicator of the presence of the gene of interest.Particularly readily detectable amplification products are betweenapproximately 50 and 250 bp. Even more preferably, amplification usingthe at least one primer pair for amplification of the methylated orunmethylated gene of interest produces an amplification product ofbetween approximately 100 and 200 bp or between 50 and 100 bp. This isparticularly relevant for tissue samples, especially paraffin embeddedsamples where limited DNA quality is typically obtained and smalleramplicons may be desired. In a preferred embodiment, the detectableamplification product comprises at least the nucleotide sequence of anyof SEQ ID NO. 2, 4, 5, 7, 8, 11, 13, 14, 16, 17 or 19. Preferably, anamplification product of (around) 100 bp, 110 bp, 115 bp, 120 bp, 125bp, 126 bp, 130 bp, 135 bp, 140 bp or 142 bp is produced.

At least one primer in the primer pair, and preferably both primers, isdesigned to bind only to the sequence of methylated or unmethylated DNAfollowing treatment with the reagent. Thus, the primer acts todiscriminate between a methylated and an unmethylated gene by basepairing only with the either the methylated form of the gene (whichremains unmodified following treatment with the reagent) or theunmethylated form of the gene (which is modified by the reagent)depending upon the application to which the methods are put. The primermust, therefore, cover at least one methylation site in the gene ofinterest. Preferably, the primer binds to a region of the gene includingat least 1, 2, 3, 4, 5, 6, 7 or 8 methylation sites. Most preferably theprimer is designed to bind to a sequence in which all cytosine residuesin CpG pairs within the primer binding site are methylated orunmethylated—i.e. a “fully methylated” or a “fully unmethylated”sequence. However, if only a single or a few methylation sites are offunctional relevance, the primer may be designed to bind to a targetsequence in which only these residues must be methylated (remain as acytosine) or unmethylated (converted to uracil) for effective binding totake place. Other (non-functionally relevant) potential sites ofmethylation may be avoided entirely through appropriate primer design orprimers may be designed which bind independently of the methylationstatus of these less relevant sites (for example by including a mix of Gand A residues at the appropriate location within the primer sequence).Accordingly, an amplification product is expected only if the methylatedor unmethylated form of the gene of interest was present in the originalDNA-containing sample. Additionally or alternatively, it may beappropriate for at least one primer in the primer pair to bind only tothe sequence of unmethylated DNA following treatment with the reagent,and the other primer to bind to methylated DNA only followingtreatment—for example where a gene involves functionally important siteswhich are methylated and separate functionally important sites which areunmethylated.

Preferably, at least one primer in the primer pair is a primercontaining a stem loop or “hairpin” structure carrying a donor and anacceptor moiety of a molecular energy transfer pair. This primer may ormay not be a primer which discriminates between methylated andunmethylated DNA as desired. The primer is arranged such that in theabsence of amplification, the acceptor moiety quenches fluorescenceemitted by the donor moiety upon excitation. Thus, prior to, or in theabsence of, amplification directed by the primer the stem loop or“hairpin” structure remains intact. Fluorescence emitted by the donormoiety is effectively accepted by the acceptor moiety leading toquenching of fluorescence.

During amplification, the configuration of the stem loop or hairpinstructure of the primer is altered. In particular, once the primer isincorporated into an amplification product, and in particular into adouble stranded DNA, (particularly during the second round ofamplification) the stem loop or hairpin structure is disrupted. Thisalteration in structure separates the donor and acceptor moietiessufficiently that the acceptor moiety is no longer capable ofeffectively quenching the fluorescence emitted by the donor moiety.Thus, the donor moiety produces a detectable fluorescence signal. Thissignal is detected in real-time to provide an indication of the genecopy number of the methylated or unmethylated gene of interest.

Thus, the methods of the invention may utilise oligonucleotides foramplification of nucleic acids that are detectably labelled withmolecular energy transfer (MET) labels. The primers contain a donorand/or acceptor moiety of a MET pair and are incorporated into theamplified product of an amplification reaction, such that the amplifiedproduct contains both a donor and acceptor moiety of a MET pair.

When the amplified product is double stranded, the MET pair incorporatedinto the amplified product may be on the same strand or, when theamplification is triamplification, on opposite strands. In certaininstances wherein the polymerase used in amplification has 5′-3′exonuclease activity, one of the MET pair moieties may be cleaved fromat least some of the population of amplified product by this exonucleaseactivity. Such exonuclease activity is not detrimental to theamplification methods of the invention.

The methods of the invention, as discussed herein are adaptable to manymethods for amplification of nucleic acid sequences, includingpolymerase chain reaction (PCR), triamplification, and otheramplification systems.

In a preferred embodiment, the MET is fluorescence resonance energytransfer (FRET), in which the oligonucleotides are labelled with donorand acceptor moieties, wherein the donor moiety is a fluorophore and theacceptor moiety may be a fluorophore, such that fluorescent energyemitted by the donor moiety is absorbed by the acceptor moiety. Theacceptor moiety may be a quencher. Thus, the amplification primer is ahairpin primer that contains both donor and acceptor moieties, and isconfigured such that the acceptor moiety quenches the fluorescence ofthe donor. When the primer is incorporated into the amplificationproduct its configuration changes, quenching is eliminated, and thefluorescence of the donor moiety may be detected.

The methods of the invention permit detection of an amplificationproduct without prior separation of unincorporated oligonucleotides.Moreover, they allow detection of the amplification product directly, byincorporating the labelled oligonucleotide into the product.

In a preferred embodiment, the methods of the invention also involvedetermining the expression of a reference gene. Reference genes areimportant to allow comparisons to be made between different samples. Byselecting an appropriate gene believed to be expressed in a stable andreliable fashion between the samples to be compared, detectingamplification of a reference gene together with the gene of interesttakes into account inter-sample variability, such as amount of inputmaterial, enzymatic efficiency, sample degradation etc. A reference geneshould ideally, in the presence of a reliable amount of input DNA, beone which is constantly expressed between the samples under test. Thus,the results from the gene of interest can be normalised against thecorresponding copy number of the reference gene. Suitable referencegenes for the present invention include beta-actin,glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal RNA genessuch as 18S ribosomal RNA and RNA polymerase II gene (Radonic A. et al.,Biochem Biophys Res Commun. 2004 Jan. 23; 313 (4) : 856-62). In aparticularly preferred embodiment, the reference gene is beta-actin.

Thus the methods of the invention may be further characterised inamplifying at least a portion of a reference gene using at least oneprimer pair, wherein at least one primer in the primer pair is a primercontaining a stem loop structure having the aforementionedcharacteristics.

Any suitable portion of the reference gene may be amplified according tothe methods of the invention, provided that the amplification product isdetectable as a reliable indicator of the presence of the reference-gene. Particularly readily detectable amplification products arebetween approximately 50 and 250 bp. Even more preferably, amplificationusing the at least one primer pair for amplification of the referencegene produces an amplification product of between approximately 100 and200 bp. This is particularly relevant for tissue samples, especiallyparaffin embedded samples where limited DNA quality is typicallyobtained.

In the embodiments in which a reference gene is included in the methodsof the invention the methods may be further characterised in that thestep of the methods which comprises quantifying the results of the(real-time) detection against a standard curve for the methylated orunmethylated gene of interest also comprises quantifying the results ofthe real-time detection of the reference gene against a standard curvefor the reference gene to produce an output, of gene copy number in eachcase and optionally further comprises normalising the results bydividing the gene copy number of the methylated or unmethylated gene ofinterest by the gene copy number of the reference gene.

Again, the methods are characterised in that the amplification isconsidered valid where the cycle threshold value is less than 40. Thisis preferably the case for both the gene of interest and reference gene.

Amplification of at least a portion of the reference gene generallyutilises at least one primer pair. Preferably, at least one primer inthe primer pair is a primer containing a stem loop structure carrying adonor and an acceptor moiety of a molecular energy transfer pair, as forthe gene of interest. The mode of action of such structure duringamplification, has been explained herein.

The “hairpin” primers for use in the methods of the invention are mostpreferably as described in U.S. Pat. No. 6,090,552 and EP 0912597, thedisclosures of which are hereby incorporated in their entirety. Theseprimers are commercially known as Amplifluor® primers. Thus, in aparticularly preferred embodiment, the primer containing a stem loopstructure used to amplify a portion of the gene of interest and/orreference gene comprises, consists essentially of or consists of thefollowing contiguous sequences in 5′ to 3′ order:

-   (a) a first nucleotide sequence of between approximately 6 and 30    nucleotides, wherein a nucleotide within said first nucleotide    sequence is labelled with a first moiety selected from the donor    moiety and the acceptor moiety of a molecular energy transfer pair,    wherein the donor moiety emits fluorescence at one or more    particular wavelengths when excited, and the acceptor moiety absorbs    and/or quenches said fluorescence emitted by said donor moiety;-   (b) a second, single-stranded nucleotide sequence comprising,    consisting essentially of or consisting of between approximately 3    and 20 nucleotides;-   (c) a third nucleotide sequence comprising, consisting essentially    of or consisting of between approximately 6 and 30 nucleotides,    wherein a nucleotide within said third nucleotide sequence is    labelled with a second moiety selected from said donor moiety and    said acceptor moiety, and said second moiety is the member of said    group not labelling said first nucleotide sequence, wherein said    third nucleotide sequence is complementary in reverse order to said    first nucleotide sequence such that a duplex can form between said    first nucleotide sequence and said third nucleotide sequence such    that said first moiety and second moiety are in proximity such that,    when the donor moiety is excited and emits fluorescence, the    acceptor moiety absorbs and quenches said fluorescence emitted by    said donor moiety; and

(d) at the 3′ end of the primer, a fourth, single-stranded nucleotidesequence comprising, consisting essentially of or consisting of betweenapproximately 8 and 40 nucleotides that comprises at its 3′ end asequence of any of SEQ ID NO. 2, 4, 5, 7, 8, 11, 13, 14, 16, 17, 19 or25 (and thus able to prime synthesis by a nucleic acid polymerase of anucleotide sequence complementary to a nucleic acid strand comprisingthe portion of the methylated or unmethylated DNA of the gene); whereinwhen said duplex is not formed, said first moiety and said second moietyare separated by a distance that, prevents molecular energy transferbetween said first and second moiety.

In a particularly preferred embodiment, the donor moiety and acceptormoiety form a fluorescence resonance energy transfer (FRET) pair.Molecular energy transfer (MET) is a process by which energy is passednon-radiatively between a donor molecule and an acceptor molecule.Fluorescence resonance energy transfer (FRET) is a form of MET. FRETarises from the properties of certain chemical compounds; when excitedby exposure to particular wavelengths of light, they emit light (i.e.,they fluoresce) at a different wavelength. Such compounds are termedfluorophores. In FRET, energy is passed non-radiatively over a longdistance (10-100 Å) between a donor molecule, which is a fluorophore,and an acceptor molecule. The donor absorbs a photon and transfers thisenergy nonradiatively to the acceptor (Förster, 1949, Z. Naturforsch.A4: 321-327; Clegg, 1992, Methods Enzymol. 211: 353-388). When twofluorophores whose excitation and emission spectra overlap are in closeproximity, excitation of one fluorophore will cause it to emit light atwavelengths that are absorbed by and that stimulate the secondfluorophore, causing it in turn to fluoresce. In other words, theexcited-state energy of the first (donor) fluorophore is transferred bya resonance induced dipole-dipole interaction to the neighbouring second(acceptor) fluorophore. As a result, the lifetime of the donor moleculeis decreased and its fluorescence is quenched, while the fluorescenceintensity or the acceptor molecule is enhanced and depolarized. When theexcited-state energy of the donor is transferred to a non-fluorophoreacceptor, the fluorescence of the donor is quenched without subsequentemission of fluorescence by the acceptor. In this case, the acceptorfunctions as a quencher. Both quenchers and acceptors may be utilised inthe present invention. Pairs of molecules that can engage influorescence resonance energy transfer (FRET) are termed FRET pairs. Inorder for energy transfer to occur, the donor and acceptor moleculesmust typically be in close proximity (up to 70 to 100 Å) (Clegg, 1992,Methods Enzymol. 211: 353-388; Selvin, 1995, Methods Enzymol. 246:300-334). The efficiency of energy transfer falls off rapidly with thedistance between the donor and acceptor molecules. According to Förster(1949, Z. Naturforsch. A4:321-327), the efficiency of energy transfer isproportional to D×10⁻⁶, where D is the distance between the donor andacceptor. Effectively, this means that FRET can most efficiently occurup to distances of about 70 Å. Molecules that are commonly used in FRETare discussed in a separate section. Whether a fluorophore is a donor oran acceptor is defined by its excitation and emission spectra, and thefluorophore with which it is paired. For example, FAM is mostefficiently excited by light with a wavelength of 488 nm, and emitslight with a spectrum of 500 to 650 nm, and an emission maximum of 525nm. FAM is a suitable donor fluorophore for use with JOE, TAMRA, and ROX(all of which have their excitation maximum at 514 nm).

In one particularly preferred embodiment, said donor moiety isfluorescein or a derivative thereof, and said acceptor moiety is DABCYL.Preferably, the fluorescein derivative comprises, consists essentiallyof or consists of 6-carboxy fluorescein.

The MET labels can be attached at any suitable point in the primers. Ina particularly preferred embodiment, the donor and acceptor moieties arepositioned on complementary nucleotides within the stem loop structure,such that whilst the stem loop is intact, the moieties are in closephysical proximity to one another. However, the primers of the inventionmay be labelled with the moieties in any position effective to allowMET/FRET between the respective donor and acceptor in the absence ofamplification and separation of the donor and acceptor once the primeris incorporated into an amplification product.

The stem loop or hairpin structure sequence does not depend upon thenucleotide sequence of the target gene (gene of interest or referencegene) since it does not bind thereto. Accordingly, “universal” stem loopor hairpin sequences may be designed which can then be combined with asequence specific primer to facilitate real-time detection of a sequenceof interest. The main sequence requirement is that the sequence forms astem loop/hairpin structure which is stable in the absence ofamplification (and thus ensures efficient quenching). Thus, the sequencespecific portion of the primer binds to a template strand and directssynthesis of the complementary strand. The primer therefore becomes partof the amplification product in the first round of amplification. Whenthe complimentary strand is synthesised, amplification occurs throughthe stem loop/hairpin structure. This separates the fluorophore andquencher molecules, thus leading to generation of florescence asamplification proceeds.

The stem loop structure is preferably found at the 5′ end of thesequence specific portion of the primer used in the amplification.

As mentioned above, this detector sequence is generally labelled with aFRET pair. Preferably, one moiety in the FRET pair is found towards,near or at the 5′end of the sequence and the other moiety is foundtowards, near or at the 3′end of the sequence such that, when the stemloop or hairpin structure remains intact FRET is effective between thetwo moieties.

As detailed in the experimental section, primers must be carefullyselected in order to ensure sensitivity and specificity of the methodsof the invention. Accordingly, particularly preferred primers for use indetecting methylation status of the gene include a primer comprising,consisting essentially of or consisting of the nucleotide sequence setforth as:

(SEQ ID NO: 1) 5′-AGCGATGCGTTCGAGCATCGCU-3′ (SEQ ID NO. 2)5′-ATTTTTGTTTGGAATTTAGGGTAG-3′ and/or, (SEQ ID NO. 3)5′-AGCGATGCGTTCGAGCATCGCUCCAACATCAAACCATCACTCA-3′ and/or, (SEQ ID NO. 4)5′-CCAACATCAAACCATCACTCA-3′ and/or, (SEQ ID NO. 5)5′-TGGAATTTAGGGTAGTATTGT-3′ and/or, (SEQ ID NO. 6)5′-AGCGATGCGTTCGAGCATCGCUTGGAATTTAGGGTAGTATTGT-3′ and/or, (SEQ ID NO. 7)5′-CCCTCCACCAACATCAAA-3′ and/or, (SEQ ID NO. 8)5′-TTAGGATGTGATGTTATTGATTTGT-3′ and/or, (SEQ ID NO. 9)5′-AGCGATGCGTTCGAGCATCGCUTTAGGATGTGATGTTATTGATTTG T-3′ and/or,(SEQ ID NO. 11) 5′-TGTTTGGAATTTAGGGTAGTATTGT-3′ and/or, (SEQ ID NO. 12)5′-AGCGATGCGTTCGAGCATCGCUTGTTTGGAATTTAGGGTAGTATTG T-3′ and/or,(SEQ ID NO. 13) 5′-CCATCACTCATTACTCAAAACAAA-3′ and/or, (SEQ ID NO. 14)5′-ATTTTTGTTCGGAATTTAGGGTAG-3′ and/or, (SEQ ID NO. 15)5′-AGCGATGCGTTCGAGCATCGCUCCGACGTCAAACCGTCGCTCG-3′ and/or,(SEQ ID NO. 16) 5′-CCGACGTCAAACCGTCGCTCG-3′ and/or, (SEQ ID NO. 17)5′-CGGAATTTAGGGTAGTATCGT-3′ and/or, (SEQ ID NO. 18)5′-AGCGATGCGTTCGAGCATCGCUCCCTCCGCCGACGTCAAA-3′ and/or, (SEQ ID NO. 19)5′-CCCTCCGCCGACGTCAAA-3′

-   SEQ ID NO 1 represents the sequence of the hairpin structure-   SEQ ID NO 2, 5, 8, or 11 represent forward primer sequences    complementary to the bisulfite converted unmethylated sequence of    the Mage promoter-   SEQ ID NO 1 represents the hairpin structure sequence-   SEQ ID NO 9 and 12 comprise the hairpin structure sequence and the    sequence of SEQ ID NO. 5, 8 and 11 respectively.-   SEQ ID NO 4, 7 and 13, represent the reverse primer sequence    complementary to the bisulfite converted unmethylated sequence of    the Mage promoter.-   SEQ ID NO 3 comprises the hairpin structure sequence and the    sequence of SEQ ID NO. 4.-   SEQ ID NO 14 and 17 represent forward primer sequences complementary    to the bisulfite converted methylated sequence of the Mage promoter-   SEQ ID NO 16 and 19, represent the reverse primer sequence    complementary to the bisulfite converted methylated sequence of the    Mage promoter.-   SEQ ID NO 15 and 18 comprises the hairpin structure sequence and the    sequence of SEQ ID NO. 16 and 19 respectively.

As detailed in the experimental section, expression and methylationlevels of MAGE-A3 showed best concordance in the assays thatincorporated SEQ ID NO. 2, 5 or 11, all three primers comprising thesequence 5′ TGGAATTTAGGGTAG 3′ (SEQ ID NO. 25). Thus in anotherembodiment, preferred primer binding to the promoter region of MAGE-A3comprises SEQ ID NO. 25. The part of the primer complementary to thebisulfite converted sequence of the MAGE-A3 is preferably less than 25bp; it is preferably 23, 22, 21, 20 or 19 bp in length. Thus the Mage-A3specific part of such preferred primer is preferably between 24 and 18bp, or between 23 and 19 bp in length. Preferably it is 19 bp in length.The primer may thus comprise any sequence of 23, 22, 21, 20, or 19consecutive bases from the sequence 5′-ATTTTTGTTTGGAATTTAGGGTAGTATTGT-3′(SEQ ID NO. 26), The Mage-A3 specific part of the primer most preferablyconsists of the nucleotide sequence set forth as SEQ ID No. 2, 4, 5 or7.

A primer comprising, consisting essentially of or consisting of thenucleotide sequence of any SEQ ID NO, 1, 3, 4, 5, 6, 7, 8, 9, 11, 12 or13 is particularly useful for the detection of hypomethylated(unmethylated) MAGE-A3 gene. Preferred primers have the nucleotidesequence of SEQ ID NO. 2, 3, 4, 5, 6 or 7.

A primer comprising, consisting essentially of or consisting of thenucleotide sequence of any SEQ ID NO. 14, 15, 16, 17, 18 or 19 isparticularly useful for the detection of hypermethylated (methylated)MAGE-A3 gene.

Preferred primer pairs for use in the methods/kits and assays of presentinvention comprise at least one primer comprising, consistingessentially of or consisting of the nucleotide sequence of any SEQ IDNO. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 25.Preferred primer pairs comprise, consist essentially of or consist ofthe nucleotide sequence of any SEQ ID NO. 2 and 3; SEQ ID NO. 6 and 7;SEQ ID NO. 9 and 4; SEQ ID NO. 12 and 13; SEQ ID NO. 14 and 15; or SEQID NO. 17 and 18. A most preferred primer pair comprises, consistsessentially of or consists of the nucleotide sequence of SEQ ID NO. 6and 7.

Either one or both of the primers may be labelled with or synthesised toincorporate a suitable stem loop or hairpin structure carrying a donorand acceptor moiety, preferably at the 5′ end, as discussed in detailabove. In a preferred embodiment, one or both of the primer(s) islabelled with or synthesised to incorporate, preferably at the 5′ end,the stem loop structure comprising, consisting essentially of orconsisting of the nucleotide sequence set forth as

(SEQ ID NO: 1) 5′-AGCGATGCGTTCGAGCATCGCU-3′.

This detector sequence is generally labelled with a FRET pair.Preferably, one moiety in the FRET pair is found towards, near or at the5′end of the sequence and the other moiety is found towards, near or atthe 3′end of the sequence such that, when the stem loop or hairpinstructure remains intact FRET is effective between the two moieties. Ina particularly preferred embodiment, the stem loop or hairpin structure,especially the nucleic acid comprising, consisting essentially of orconsisting of the sequence set forth as SEQ ID NO: 1, is labelled at the5′end with FAM and at the 3′end with DABCYL. Other preferredcombinations are discussed herein, which discussion applies mutatismutandis.

These primers form separate aspects of the present invention. Furthercharacteristics of these primers are summarized in the detaileddescription (experimental part) below. It is noted that variants ofthese sequences may be utilised in the present invention. In particular,additional flanking sequences may be added, for example to improvebinding specificity or the formation of a stem loop, as required.Variant sequences preferably have at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% nucleotide sequence identity with thenucleotide sequences of the primers and/or probes set forth in SEQ IDNO:1 to 9 and 11 to 19 or 25. The primers and hairpin structures mayincorporate synthetic nucleotide analogues as appropriate or may be DNA,RNA or PNA based for example, or mixtures thereof. Similarly alternativefluorescent donor and acceptor moieties/FRET pairs may be utilised asappropriate. In addition to being labelled with the fluorescent donorand acceptor moieties, the primers may include modified oligonucleotidesand other appending groups and labels provided that the functionality asa primer and/or stem loop/hairpin structure in the methods of theinvention is not compromised.

For each primer pair at least one primer is labelled with a donor and anacceptor moiety of a molecular energy transfer pair arranged such thatin the absence of amplification, the acceptor moiety quenchesfluorescence emitted by the donor moiety (upon excitation) and duringamplification, the stem loop structure is disrupted so as to separatethe donor and acceptor moieties sufficiently to produce a detectablefluorescence signal which is detected in real-time to provide anindication of the gene copy number of the gene. Preferably, said donormoiety and said acceptor moiety are a FRET pair. In one embodiment, saiddonor moiety and said acceptor moiety are selected from5-carboxyfluorescein or 6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′-tetramethyl-6-carboxyrhodamine (TAMRA),6-carboxy-X-rhodamine (ROX), 5-(2′-aminoethyl)aminonapthalene-1-sulfonicacid (EDANS), anthranilamide, coumarin, terbium chelate derivatives.Malachite green, Reactive Red 4, DABCYL, tetramethyl rhodamine, pyrenebutyrate, eosine nitrotyrosine, ethidium, and Texas Red. In a furtherembodiment, said donor moiety is selected from fluorescein,5-carboxyfluorescein or 6-carboxyfluorescein (FAM), rhodamine,5-(2′-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS),anthranilamide, coumarin, terbium chelate derivatives, Malachite green,and Reactive Red 4, and said acceptor moiety is selected from DABCYL,rhodamine, tetramethyl rhodamine, pyrene butyrate, eosine nitrotyrosine,ethidium, and Texas Red. Preferably, said donor moiety is fluorescein ora derivative thereof, and said acceptor moiety is DABCYL and mostpreferably the donor moiety is 6-carboxyfluorescein. Other preferredcombinations, particularly in a multiplexing context, are discussedherein and these combinations are also envisaged for these aspects ofthe invention.

The invention also provides kits which may be used in order to carry outthe methods of the invention. The kits may incorporate any of thepreferred features mentioned in connection with the various methods (anduses) of the invention described herein. Thus, the invention provides akit for detecting the presence and/or amount of a methylated orunmethylated gene of interest in a DNA-containing sample, comprising atleast one primer pair of the invention. Preferably, the kit incorporatesa primer pair of the invention for detecting the presence and/or amountof unmethylated and/or methylated MAGE-A3 gene and a primer pair fordetecting the presence and/or amount of a reference gene, in particularbeta-actin. Thus, the kit may comprise primer pairs comprising a primercomprising, consisting essentially of or consisting of the nucleotidesequence set forth as SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14,15, 16, 17, 18, 19 or 25. Preferably, at least one primer in each primerpair is labelled with an appropriate stem loop or hairpin structure tofacilitate detection in real-time, as discussed above (which discussionapplies here mutatis mutandis). Most preferably at least one primer ineach primer pair incorporates the stem loop or hairpin Structure whichcomprises, consists essentially of or consists of the nucleotidesequence set forth as SEQ ID NO:1. The stem loop structure is labelledwith an appropriate donor and acceptor moiety, as discussed herein(which discussion applies here mutatis mutandis).

As aforementioned, further characteristics of the primers of theinvention are summarized in the detailed description (experimental part)below. Variants of these sequences may be utilised in the presentinvention as discussed herein. Alternative fluorescent donor andacceptor moieties/FRET pairs may be utilised as appropriate, asdiscussed herein.

In one embodiment, the kit of the invention further comprises a reagentwhich modifies unmethylated cytosine, as discussed herein (in preferenceto methylated cytosine residues which are protected). Such a reagent isuseful for distinguishing methylated from unmethylated cytosineresidues. In a preferred embodiment, the reagent comprises bisulphite,preferably sodium bisulphite. This reagent is capable of convertingunmethylated cytosine residues to uracil, whereas methylated cytosinesremain unconverted. This difference in residue may be utilised todistinguish between methylated, and unmethylated nucleic acid in adownstream process, such as PCR using primers which distinguish betweencytosine and uracil (cytosine pairs with guanine, whereas uracil pairswith adenine).

As discussed with respect to the methods of the invention herein,suitable controls may be utilised in order to act as quality control forthe methods. Accordingly, in one embodiment, the kit of the inventionfurther comprises, consists essentially of or consists of one or morecontrol nucleic acid molecules of which the methylation status is known.These (one or more) control nucleic acid molecules may include bothnucleic acids which are known to be, or treated so as to be, methylatedand/or nucleic acid molecules which are known to be, or treated so as tobe, unmethylated. One example of a suitable internal reference gene,which is generally unmethylated, but may be treated so as to bemethylated, is beta-actin.

The kits of the invention may additionally include suitable buffers andother reagents for carrying out the claimed methods of the invention.Thus, the discussion provided in respect of the methods of the inventionapplies mutatis mutandis here and is not repeated for reasons ofconciseness. In one embodiment, the kit of the invention furthercomprises, consists essentially of, or consists of nucleic acidamplification buffers.

The kit may also additionally comprise, consist essentially of orconsist of enzymes to catalyze nucleic acid amplification. Thus, the kitmay also additionally comprise, consist essentially of or consist of asuitable polymerase for nucleic acid amplification. Examples includethose from both family A and family B type polymerases, such as Taq,Pfu, Vent etc.

The various components of the kit may foe packaged separately inindividual compartments or may, for example be stored together whereappropriate.

The kit may also incorporate suitable instructions for use, which may beprinted on a separate sheet or incorporated into the kit's packaging forexample. The instructions may facilitate use of the kits of theinvention with an appropriate real-time amplification apparatus, anumber of which are commercially available.

The last step of the real-time methods of the invention involvesquantifying the results of the real-time detection against a standardcurve for the methylated or unmethylated gene of interest, andoptionally the reference gene (where included . Standard curves may begenerated using a set of standards. Each standard contains a known copynumber, or concentration, of the gene of interest and/or reference geneas appropriate. Typically, a baseline value of fluorescence will be setto account for background fluorescence. For example, in one embodimentthe Sequence Detection System (SDS) software is utilised. This softwaresets a default baseline range of cycles 3 to 15 of the amplificationreaction before amplification products are detected. A threshold valueof fluorescence is then defined at a statistically significant valueabove this baseline. Typically, the threshold is set to 10 standarddeviations above the baseline fluorescence. Appropriate software isprovided with apparatus for carrying out real-time amplificationreactions. The software automatically calculates the baseline andthreshold values for the reaction. The threshold cycle value (Ct) canthen be determined for each standard. This is the number of cyclesrequired to achieve the threshold amplification level. Thus, the greaterthe initial concentration of the gene standard in the reaction mixture,the fewer the number of cycles required to achieve a particular yield ofamplified product. A plot of Ct against the log₁₀ of the known initialcopy number of the set of standard DNAs produces a straight line. Thisis the standard curve. Thus, the Ct value for the amplification of thegene of interest and reference gene, where utilised, can each beinterpolated against the respective standard curve in order to determinethe copy number in the DNA-containing sample. Thus, the output of themethod is the gene copy number for each of the gene of interest andreference gene. The results may be normalised by dividing the gene copynumber of the methylated or unmethylated gene of interest by the genecopy number of the reference gene. In a preferred embodiment, theApplied Biosystems 7900 HT fast real-time PCR system is used to carryout the methods of the invention. Preferably, SDS software is utilised,preferably including a suitable algorithm such as the Auto CT algorithmfor automatically generating baseline and threshold values forindividual detectors.

Whilst the methods of the invention may be utilised with any suitableamplification technique, it is most preferred that amplification iscarried out using the polymerase chain reaction (PCR). Thus, whilst PCRis a preferred amplification method, to include variants on the basictechnique such as nested PCR, equivalents may also be included withinthe scope of the invention. Examples include, without limitation,isothermal amplification techniques seen as NASBA, 3SR, TMA andtriamplification, all of which are well known in the art and suitablereagents are commercially available. Other suitable amplificationmethods include, without limitation, the ligase chain reaction (LCR)(Barringer et al, 1990), MLPA, selective amplification of targetpolynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequenceprimed polymerase chain reaction (U.S. Pat. No. 4,437,975), invadertechnology (Third Wave Technologies, Madison, Wis.), strand displacementtechnology, arbitrarily primed polymerase chain reaction (WO90/06995)and nick displacement amplification (WO2004/067726).

The real-time PCR methods of the invention generally involve steps oflowering the temperature to allow primer annealing, raising thetemperature for primer extension, raising the temperature fordenaturation and lowering the temperature for data-collection. In onespecific embodiment, the data-collection step is carried out at atemperature of between approximately 60° C. and 64° C., most preferablyat approximately 62° C. since this has been shown to give maximallysensitive and specific results as discussed in Example section.

In a specific embodiment, the thermal profiling of the polymerase chainreaction comprises between 40 and 50 repeats, preferably approximately45 repeats of the cycle:

-   (a) approximately 50° C. for approximately 2 minutes-   (b) approximately 95° C. for approximately 10 minutes-   (c) approximately 95° C. for approximately 15 seconds-   (d) approximately 62° C. for approximately 1 minute

The preferred reaction scheme shown to produce specific and sensitiveresults in the methods of the invention is Stage1: 50° C. for 2 min,Stage2: 95° C. for 10 min, Stage3: 95° C. for 15 sec, 59° C. for 30 sec,59° C. for 30 sec (=plateau−data collection) for 45 repeats.

It is possible for the methods of the invention to be used in order todetect more than one gene of interest in the same reaction. Through theuse of several specific sets of primers, amplification of severalnucleic acid targets can be performed in the same reaction mixture. Thismay be termed “multiplexing”. In a preferred embodiment, one or bothprimers for each target, may foe hairpin primers labeled with afluorescent moiety and a quenching moiety that form a FRET pair.Amplification of several nucleic acid targets requires that a differentfluorescent donor and/or acceptor moiety, with a different emissionwavelength, be used to label each set of primers. During detection andanalysis after an amplification, the reaction mixture is illuminated andread at each of the specific wavelengths characteristic for each of thesets of primers used in the reaction. It can thus be determined whichspecific target DNAs in the mixture were amplified and labelled. In aspecific embodiment, two or more primer pairs for amplification ofdifferent respective target sequences are used. Thus the presence and/oramount of a panel of methylated/unmethylated genes of interest can bedetected in a single DNA-containing sample

Multiplexing can also be utilised in the context of detecting both thegene of interest and a reference gene in the same reaction. Again,primers labelled with appropriate distinguishable donor and/or acceptormoieties allow the signal generated by amplification of the gene ofinterest and reference gene respectively to be distinguished.

In one embodiment, a universal quencher is utilised together withsuitable fluorophore donors each having a distinguishable emissionwavelength maximum. A particularly preferred quencher is DABCYL.Together with DABCYL as quencher, the following fluorophores may each beutilised to allow multiplexing: Coumarin (emission maximum of 475 nm),EDANS (491 nm), fluorescein (515 nm), Lucifer yellow (523 nm), BODIPY(525 um), Eosine (543 nm), tetramethylrhodamine (575 nm) and texas red(615 nm) (Tyagi et al., Nature Biotechnology, Vol. 16, January 1998;43-53). Other preferred combinations are discussed herein.

In an alternative embodiment, the DNA containing sample can be split andthe methods of the invention carried out on suitable portions of thesample in order to obtain directly comparable results. Thus, where boththe gene of interest and a reference gene are detected, the sample maybe split two ways to allow detection of amplification of the gene ofinterest in real time in one sample portion and detection ofamplification of the reference gene in real time in the other sampleportion. The sample may be split further to allow suitable controlreactions to be carried out, as required. The benefit of this scheme isthat a universal FRET pair can be used to label each primer pair andremoves the requirement to detect emission at a range of wavelengths.However, this method does rely upon obtaining a suitable sampleinitially to permit dividing the sample. Whilst any suitable reactionvolume may be utilised, in one specific embodiment, the total reactionvolume for the amplification step is between approximately 10 and 40 μl,more preferably between approximately 10 and 30 μl and most preferablyaround 12 μl

In one aspect, the oligonucleotides, primers or probes, primer pairs,kits or methods of the present invention are used for diagnosing canceror predisposition of cancer, wherein the presence of unmethylated (orhypomethylated) MAGE-A3 in the sample is indicative for cancer orpredisposition to cancer. Thus, the present invention provides kits,methods and primers for diagnosing cancer or predisposition to cancer.

“Diagnosis” is defined herein to include screening for a disease orpre-stadia of a disease, identifying a disease or prestadia of adisease, monitoring staging and the state and progression of thedisease, checking for recurrence of disease following treatment andmonitoring the success of a particular treatment. The tests may alsohave prognostic value, and this is included within the definition of theterm “diagnosis”. The prognostic value of the tests may be used as amarker of potential susceptibility to cancer or as a marker forprogression to cancer. Thus patients at risk may be identified beforethe disease has a chance to manifest itself in terms of symptomsidentifiable in the patient. In a preferred embodiment, the cancer isselected from lung cancer, melanoma or bladder cancer. In a preferredembodiment, the methods and assays for diagnosis use at least oneoligonucleotide comprising, consisting, consisting essentially of, orconsisting of the nucleotide sequence of any SEQ ID NO. 2, 3, 4, 5, 6,7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 25. In a preferredembodiment, diagnosis of cancer or predisposition to cancer usesoligonucleotides comprising, consisting, consisting essentially of, orconsisting of the nucleotide sequence of any SEQ ID NO. 2, 3, 4, 5, 6,7, 8, 9, 11, 12, 13, or 25 and detects the unmethylated form of thegene. In an alternative embodiment, the methods and assays for diagnosisUse at least one oligonucleotide comprising, consisting essentially of,or consisting of the nucleotide sequence of any SEQ ID NO. 14, 16, 17 or19.

Testing can be performed diagnostically or in conjunction with atherapeutic regimen. As mentioned above, RT-PCR assays that establishthe predictive value of MAGE-A3 expression in NSCLC have been described.These assays find their application in the selection of patientssuitable for treatment with a MAGE-A3 immunotherapeutic. The inventorshave shown that an assay designed for the detection of unmethylatedMAGE-A3 employing oligonucleotides, primers or probes, primer pairs orkits of the invention, can reliably categorize samples as MAGE-A3expressing. The methylation status result obtained with the methylationtest is in good concordance with the results obtained with an existingRT-PCR test for MAGE-A3 detection that is used on RNA samples.Accordingly, the methylation test has clinical application.

In a further aspect the invention provides a method of predicting thelikelihood of successful treatment of cancer in a subject comprising:

-   -   (a) contacting/treating a DNA-containing test sample obtained        from a subject with a reagent which selectively modifies        unmethylated cytosine residues in the DNA to produce detectable        modified residues but which does not modify methylated cytosine        residues    -   (b) amplifying at least a portion of the unmethylated MAGE A3        gene using at least one primer pair, at least one primer of        which is designed to bind only to the sequence of unmethylated        DNA respectively following treatment with the reagent, wherein        at least one primer in the primer pair comprises, consists        essentially of, or consists of the nucleotide sequence of any of        SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 25    -   (c) determining the methylation status of the MAGE-A3 gene;        wherein the presence of unmethylated MAGE-A3 in the sample        indicates that the likelihood of successful treatment with a        MAGE-A3 immunotherapeutic is higher than if no or lower levels        of unmethylated MAGE-A3 gene is detected.

Step (c) involves identifying whether an amplification product hasformed. The identification of the amplification product fusing anysuitable technique as discussed herein) indicates the present ofunmethylated or hypomethylated MAGEA3 in the sample.

Of course, the reverse situation is also applicable and so the methodsof the invention may likewise be utilised in order to determine whetherthere is likely to be resistance to, or unsuccessful treatment using, anMAGEA3 immunotherapeutic agent—the absence of unmethylated MAGE-A3 inthe sample indicates there is likely to be resistance to treatmentand/or that treatment is likely to be unsuccessful. Primers specific formethylated DNA may also be employed in complementary methods, in certainembodiments.

The methods of the invention may also be utilised to select a suitablecourse of treatment for a patient—the presence of unmethylated MAGE-A3indicates that MAGE-A3 immunotherapeutic agents may be beneficiallyadministered, whereas the absence or low level of unmethylated MAGE-A3indicates that immonothereapeutic agents are contra-indicated. Thediscussion provided in respect of the oligonucleotides, primers orprobes, primer pairs, kits or methods of the invention applies to thepresent aspect mutatis mutandis and all embodiments are thereforeenvisaged, as appropriate, for this aspect of the invention.

By “likelihood of successful treatment” is meant the probability thattreatment of the cancer using any one or more of the listed therapeuticagents, preferably a MAGE-A3 immunotherapeutic or a compositioncomprising MAGE-A3, will be successful.

“Resistance” is defined as a reduced probability that treatment ofcancer will be successful using any one of the specifiedimmunotherapeutic agents and/or that higher dose will be required toachieve a therapeutic effect.

Hypomethylation of MageA3 may be linked to certain cancer types.Accordingly, in a specific embodiment, the invention provides a methodof detecting a predisposition to, or the incidence of, bladder cancer,lung cancer, including NSCLC or melanoma in a sample comprisingdetecting the methylation status of the MAGE-A3 gene using theoligonucleotides, primers or probes, primer pairs, kits or methods ofthe invention, wherein detection of unmethylated MAGE-A3 in the sampleis indicative of a predisposition to, or the incidence of, cancer and inparticular melanoma; lung cancer including non-small cell lung carcinoma(NSCLC); or bladder cancer, including transitional cell carcinoma. In afurther embodiment, the tumour or cancer is selected from breast cancer;head and neck cancer including oesophagus carcinoma; squamous cellcarcinoma; seminoma; liver cancer; multiple myeloma and colon carcinoma.

In a further aspect, there is provided a method for determining thepresence Of a MAGE-A3 positive tumor comprising detecting themethylation status of the MAGE-A3 gene in a sample with use of theoligonucleotides, primers or probes, primer pairs, kits or methodsdescribed herein, wherein the presence of unmethylated MAGE-A3 isindicative for the presence of a MAGE-A3 positive tumor.

Testing can be performed diagnostically or in conjunction with atherapeutic regimen. MAGE-A3 specific immunotherapeutics (ASCI) havebeen developed and are currently being evaluated in clinical trials.Testing can also be used to determine what therapeutic or preventiveregimen to employ on a patient and be used to monitor efficacy of atherapeutic regimen.

Accordingly, the invention further provides a method for identifyingand/or selecting a patient suitable for treatment with a MAGE-A3immunotherapeutic comprising detecting the methylation status of theMAGE-A3 gene in a sample of the patient with use of theoligonucleotides, primers or probes, primer pairs, kits or methodsdescribed herein, wherein if the MAGE-A3 gene is unmethylated thesubject is identified and/or selected for treatment with the MAGE-A3immunotherapeutic.

Alternatively, if the gene is not unmethylated the subject is preferablynot selected for treatment with a MAGE-A3 immunotherapeutic.

In a related aspect, the invention provides a method for predicting thelikelihood of successful treatment of cancer comprising detecting themethylation status of the MAGE-A3 gene in a sample of the patient withuse of the oligonucleotides, primers or probes, primer pairs, kits ormethods described herein, wherein if the gene is unmethylated thelikelihood of successful treatment with a MAGE-A3 immunotherapeutic ishigher than if the gene is methylated.

Alternatively, the absence of unmethylated MAGE-A3 in the sampleindicates that the likelihood of resistance to treatment with a MAGE-A3immunotherapeutic is higher than if the gene is unmethylated. Thus, thedetection of a methylated MAGE-A3 gene (or lack of detection of thehypomethylated gene) indicates that the probability of successfultreatment with an immunotherapeutic is low.

Thus, the patient population may be selected for treatment on the basisof their methylation status with respect to the MAGE-A3 gene. This leadsto a much more focussed and personalised form of medicine and thus leadsto improved success rates since patients will be treated with drugswhich are most likely to be effective.

In a further related aspect, the invention provides a method ofselecting a suitable treatment regimen for cancer comprising detectingthe methylation status of the MAGE-A3 gene in a sample of the patientwith use of the oligonucleotides, primers or probes, primer pairs, kitsor methods described herein, wherein if the gene is unmethylated, animmunotherapeutic (in particular a MAGE immunotherapeutic) is selectedfor treatment.

Alternatively, if the gene is not unmethylated, treatment with animmunotherapeutic is contra-indicated.

Also provided is a method of treating cancer in a subject comprisingadministration of an immunotherapeutic, wherein the subject has beenselected for treatment on the basis of measuring the methylation statusof a MAGE-A3 gene, according to any of the methods of the invention orby using an oligonucleotide, primer or probe, primer pair, kit or amethod as described herein. Preferably, for all of the different aspectsdescribed herein, the detection of unmethylated MAGE-A3 gene correspondsto an increased level of MAGE-A3 protein.

MAGE-A3 immunotherapeutics, useful in the present invention, includeMAGE-A3 based compositions. Examples of compositions comprising MAGE-A3include compositions comprising full length MAGE-A3, substantiallyfull-length MAGE-A3 and fragments of MAGE-A3, for example peptides ofMAGE-A3.

Examples of peptides that may be used in the present invention includethe following MAGE-A3 peptides:

SEQ ID NO Peptide sequence SEQ ID NO: 27 FLWGPRALV SEQ ID NO: 28EVDPIGHLY SEQ ID NO: 29 MEVDPIGHLY SEQ ID NO: 30 VHFLLLKYRASEQ ID NO: 31 LVHFLLLKYR SEQ ID NO: 32 LKYRAREPVT SEQ ID NO: 33ACYEFLWGPRALVETS SEQ ID NO: 34 TQHFVQENYLEY

The MAGE protein may be full length MAGE-A3 or may comprise asubstantially full-length fragment of MAGE3, for example amino acids3-314 of MAGE3 (312 amino acids in total), or other MAGE-A3 fragments inwhich between 1 and 10 amino acids are deleted from the N-terminusand/or C-terminus of the MAGE-A3 protein.

In one embodiment, the MAGE-A3 protein, fragment or peptide may belinked to a fusion partner protein.

The MAGE-A3 protein, fragment or peptide and fusion partner protein maybe chemically conjugated, or may be expressed as a recombinant fusionprotein. In an embodiment in which the antigen and partner are expressedas a recombinant fusion protein, this may allow increased levels to beproduced in an expression system compared to non-fused protein. Thus thefusion partner protein may assist in providing T helper epitopes(immunological fusion partner protein), preferably T helper epitopesrecognised by humans, and/or assist in expressing the protein(expression enhancer protein) at higher yields than the nativerecombinant protein. In one embodiment, the fusion partner protein maybe both an immunological fusion partner protein and expression enhancingpartner protein.

In one embodiment of the invention, the immunological fusion partnerprotein that may be used is derived from protein D, a surface protein ofthe gram-negative bacterium, Haemophilus influenza B (WO 91/18926) or aderivative thereof. The protein D derivative may comprise the first ⅓ ofthe protein, or approximately the first ⅓ of the protein. In oneembodiment, the first N-terminal 109 residues of protein D may be usedas a fusion partner to provide a MAGE-A3 antigen with additionalexogenous T-cell epitopes and increase expression level in E. coli (thusacting also as an expression enhancer). In an alternative embodiment,the protein D derivative may comprise the first d-terminal 100-110 aminoacids or approximately the first N-terminal 100-110 amino acids. In oneembodiment, the protein D or derivative thereof may be lipidated andlipoprotein D may be used: the lipid tail may ensure optimalpresentation of the antigen to antigen presenting cells. In analternative embodiment, the protein D or derivative thereof is notlipidated. The “secretion sequence” or “signal sequence” of protein D,refers to approximately amino acids 1 to 16, 17, 18 or 19 of thenaturally occurring protein. In one embodiment, the secretion or signalsequence of protein D refers to the N-terminal 19 amino acids of proteinD. In one embodiment, the secretion or signal sequence is included atthe N-terminus of the protein D fusion partner. As used herein, the“first third (⅓)”, “first 109 amino acids” and “first N-terminal 100-110amino acids” refer to the amino acids of the protein D sequenceimmediately following the secretion or signal sequence. Amino acids 2-Kand 3-L of the signal sequence may optionally be substituted with theamino acids 2-M and 3-D.

In one embodiment, the MAGE-A3 may be Protein D-MAGE-A3-His, a432-amino-acid-residue fusion protein. This fusion protein comprises thesignal sequence of protein D, amino acids 1 to 109 of Protein D, 312amino acids from the MAGE-A3 protein (amino acids 3-314), a spacer and apolyhistidine tail (His) that may facilitate the purification of thefusion protein during the production process, for example:

-   i) An 18-residue signal sequence and the first N-terminal 109    residues of protein D;-   ii) Two unrelated residues (methionine and aspartic acid);-   iii) Residues 3-314 of the native MAGE-3 protein;-   iv) Two glycine residues functioning as a hinge region; and-   v) seven Histidine residues.

The amino acid sequence for this molecule is shown in FIG. 10 (SEQ IDNO: 40). This antigen and those summarised below are described in moredetail in WO 99/40188.

In another embodiment the immunological fusion partner protein may bethe protein known as LytA or a protein derived therefrom. LytA isderived from Streptococcus pneumoniae which synthesise anN-acetyl-L-alanine amidase, amidase LytA, (coded by the LytA gene (Gene,43 (1986) page 265-272)) an autolysin that specifically degrades certainbonds in the peptidoglycan backbone. The C-terminal domain of the LytAprotein is responsible for the affinity to choline or to some cholineanalogues such as DEAE. This property has been exploited for thedevelopment of E. coli C-LytA expressing plasmids useful for expressionof fusion proteins. Purification of hybrid proteins containing theC-LytA fragment at its amino terminus has been described (Biotechnology:10, (1992) page 795-798). In one embodiment, the C terminal portion ofthe molecule may be used. The repeat portion of the LytA molecule foundin the C terminal end starting at residue 178 may be utilised. In oneembodiment, the LytA portion may incorporate residues 188-305.

Other fusion partners include the non-structural protein from influenzaevirus, NS1 (hemagglutinin). In one embodiment, the N terminal 81 aminoacids of NS1 are utilised, although different fragments may be usedprovided they include T-helper epitopes.

In one embodiment of the present invention, the MAGE-A3 protein maycomprise a derivatised free thiol. Such antigens have been described inWO99/40188. In particular carboxyamidated or carboxymethylatedderivatives may be used.

In a further embodiment the MAGE-A3 composition comprises a nucleic acidmolecule encoding a MAGE-A3 protein, fragment or peptide or fusionprotein as described herein. In one embodiment of the present invention,the sequences may be inserted into a suitable expression vector and usedfor DNA/RNA vaccination. Microbial vectors expressing the nucleic acidmay also be used as vector-delivered immunotherapeutics.

Examples of suitable viral vectors include retroviral, lentiviral,adenoviral, adeno-associated viral, herpes viral including herpessimples viral, alpha-viral, pox viral such as Canarypox andvaccinia-viral based systems. Gene transfer techniques using theseviruses are Known to those skilled in the art. Retrovirus vectors forexample may be used to stably integrate the polynucleotide of theinvention into the host genome, although such recombination is notpreferred. Replication-defective adenovirus vectors by contrast remainepisomal and therefore allow transient expression. Vectors capable ofdriving expression in insect cells (for example baculovirus vectors), inhuman cells, yeast or in bacteria may be employed in order to producequantities of the MAGE-A3 protein encoded by the polynucleotides of thepresent invention, for example for use as subunit vaccines or inimmunoassays.

In a preferred embodiment the adenovirus used as a live vector is areplication defective simian adenovirus. Typically these viruses containan E1 deletion and can be grown on cell lines that are transformed withan E1 gene. Preferred Simian adenoviruses are viruses isolated fromChimpanzee. In particular C68 (also known as Pan 9) (See U.S. Pat. No.6,083,716) and Pan 5, 6 and Pan 7 (WO 03/046124) are preferred for usein the present invention. These vectors can be manipulated to insert aheterologous gene of the invention such that the gene product may beexpressed. The use, formulation and manufacture of such recombinantadenoviral vectors is set forth in detail in WO 03/046142.

Conventional recombinant techniques for obtaining nucleic acidsequences, and production of expression vectors are described inManiatis et al., Molecular Cloning—A Laboratory Manual; Cold SpringHarbor, 1982-1989.

For protein based compositions, the proteins of the present inventionmay be provided either soluble in a liquid form or in a lyophilisedform.

Each human dose may comprise 1 to 1000 μg of protein. In one embodiment,the dose may comprise 30-300 μg of protein.

The MAGE-A3 containing composition as described herein may furthercomprise a vaccine adjuvant, and/or an immunostimulatory cytokine orchemokine.

Suitable vaccine adjuvants for use in the present invention arecommercially available such as, for example, Freund's IncompleteAdjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.);Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) ; AS-2(SmithKline Beecham, Philadelphia, Pa.); aluminium salts such asaluminium hydroxide gel (alum) or aluminium phosphate; salts of calcium,iron or zinc; an insoluble suspension of acylated tyrosine; acylatedsugars; cationically or anionically derivatised polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A andquil A. Cytokines, such as GM-CSF or interleukin2, -7, or -12, andchemokines may also be used as adjuvants.

In one embodiment, the adjuvant may comprise a combination ofmonophosphoryl lipid A, such as 3-de-O-acylated monophosphoryl lipid A(3D-MPL) together with an aluminium salt. Alternatively, the adjuvantmay comprise 3D-MPL or other toll like receptor 4 (TLR4) ligands such asaminoalkyl glucosaminide phosphates as disclosed in WO 98/50399, WO01/34617 and WO 03/065806.

Another adjuvant that may be used is a saponin, for example QS21 (AquilaBiopharmaceuticals Inc., Framingham, Mass.), that may be used alone orin combination with other adjuvants. For example, in one embodiment,there is provided a combination of a monophosphoryl lipid A and saponinderivative, such as the combination of QS21and 3D-MPL as described in WO94/00153, or a composition in which the QS21 is quenched withcholesterol, as described in WO 90/33739. Other suitable formulationscomprise an oil-in-water emulsion and tocopherol. In one embodiment, theadjuvant comprises QS21, 3D-MPL, and tocopherol in an oil-in-wateremulsion, as described in WO 95/17210.

Other adjuvants for use in the present invention may comprise TLR9antagonists such as unmethylated CpG containing oligonucleotides, inwhich the CpG dinucleotide is unmethylated. Such oligonucleotides arewell known and are described in, for example WO 96/02555.

Suitable oligonucleotides for use in the present invention (in thiscontext) may include:

SEQ ID NO: 35 TCC ATG ACG TTC CTG ACG TT CpG 1826 SEQ ID NO: 36TCT CCC AGC GTG CGC CAT CpG 1758 SEQ ID NO: 37ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG SEQ ID NO: 38TCG TCG TTT TGT CGT TTT GTC GTT CpG 2006, CpG 7909 SEQ ID NO: 39TCC ATG ACG TTC CTG ATG CT CpG 1668

CpG-containing oligonucleotides may also be used alone or in combinationwith other adjuvants. For example, in one embodiment, the adjuvantcomprises a combination of a CpG-containing oligonucleotide and asaponin derivative particularly the combination of CpG and QS21 asdisclosed in WO 00/09159 and WO 00/62800.

Accordingly there is provided a composition comprising MAGE-A3 asdescribed herein, wherein the adjuvant comprises one or more of 3D-MPL,QS21, a CpG oligonucleotide, a polyethylene ether or ester or acombination of two or more of these adjuvants. The MAGE-A3 componentwithin the composition may be presented in an oil in water or a water inoil emulsion vehicle or in a liposomal formulation, in certainembodiments.

In one embodiment, the adjuvant may comprise one or more of 3D-MPL, QS21and an immunostimulatory CpG oligonucleotide. In an embodiment all threeadjuvant components are present. The components may be either presentedin a liposomal formulation or an oil in water emulsion, such asdescribed in WO 95/17210.

In another embodiment 3D MPL-and Qs21 are presented in an oil in wateremulsion, and in the absence of a CpG oligonucleotide.

The amount of 3D-MPL used is generally small, but depending on theformulation may be in the region of 1-1000 μg per dose, preferably 1-500μg per dose, and more preferably between 1 to 100 μg per dose.

The amount of CpG or immunostimulatory oligonucleotides in the adjuvantsof the present invention is generally small, but depending on theformulation may be in the region of 1-1000 μg per dose, preferably 1-500μg per dose, and more preferably between 1 to 100 μg per dose.

The amount of saponin for use in the adjuvants of the present inventionmay be in the region of 1-1000 μg per dose, preferably 1-500 μg perdose, more preferably 1-250 μg per dose, and most preferably between 1to 100 μg per dose.

The adjuvant formulations as described herein may additionally comprisean oil in water emulsion and/or tocopherol or may be formulated in aliposomal composition.

Other suitable adjuvants include Montanide ISA 720 (Seppic, France), SAP(Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), RibiDetox, RC-529 (GSK, Hamilton, Mont.) and other aminoalkyl glucosaminide4-phosphates (AGPs).

Generally, each human dose may comprise 0.1-1000 μg of antigen, forexample 0.1-500 μg, 0.1-100 μg, or 0.1 to 50 μg. An optimal amount for aparticular immunotherapeutic can be ascertained by standard studiesinvolving observation of appropriate immune responses in vaccinatedsubjects. Following an initial vaccination, subjects may receive one orseveral booster immunisation adequately spaced.

Alternatively, a composition for use in the method of the presentinvention may comprise a pharmaceutical composition comprising MAGE-A3as described herein in a pharmaceutically acceptable excipient.

The invention will now be described with respect to the followingnon-limiting examples:

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Location of the MAGEA3_U primers on the non converted sequence(FIG. 1 a—SEQ ID NO: 10) and corresponding converted sequence (FIG. 1b—SEQ ID NO: 41). MAGEA3_GO1 U primer position is boxed, MAGEA3_GO2 Uprimer position is highlighted, MAGEA3_FURUTA U primer postion is inbold, MAGEA3_QIU U primer position is underlined, The G positionindicated by

corresponds to the transcription start site.

FIG. 2: Location of the MAGEA3_GO_(—)2_U primers on the non convertedsequence (FIG. 2 a—SEQ ID NO: 10) and corresponding converted sequence(FIG. 2 b—SEQ ID NO: 41), underlined starts at the transcription startsite.

FIG. 3: Limit of detection graph.

FIG. 3 a: MAGEA3_GO_(—)2_U: input U DNA (LNCaP cells) is plotted againstCt values, 1.5 ng of U input DNA is still detectable

FIG. 3 b: MAGEA3_Furuta_U: input U DNA (Gerl cells) is plotted againstCt values, 1.5 ng of U input DNA is still detectable

FIG. 4: Schematic overview of the Amplifluor® technique. At least oneprimer (forward primer in this case) in the primer pair contains a“hairpin” structure carrying a donor (FAM) and an acceptor moiety(DABCYL) of a molecular energy transfer pair. In the absence ofamplification, fluorescence emitted by the donor moiety is effectivelyaccepted by the acceptor moiety leading to quenching of fluorescence.During amplification, the primer is incorporated into an amplificationproduct. During the second round of amplification the stem loop orhairpin structure is disrupted. The acceptor moiety is no longer capableof effectively quenching the fluorescence emitted by the donor moiety.Thus, the donor moiety produces a detectable fluorescence signal.

FIG. 5: Decision tree for sample classification (Methylated,Non-Methylated or Invalid)

FIG. 6: MAGE-A3 methylation status in melanoma samples: ReceiverOperating Characteristics (ROC) curves were calculated for the 4 MAGE-A3Unmethylated assays by plotting the true positive rate (sensitivity) infunction of the false positive rate (100-specificity).

FIG. 6 a: GO_(—)1_U assay: sensitivity 91.7%, specificity 76.5%, cut-off214.8, Area under the curve (AUC) is 0.912. The 95% CI range was 0.781to 0.977 at a significance of P=0.0001 for area=5.

FIG. 6 b: GO_(—)2_U assay: sensitivity 87.5%, specificity 100%, cut-off292.6, Area under the curve (AUC) is 0.971. The 95% CI range was 0.863to 0.996 at a significance of P=0.0001 for area=5.

FIG. 6 c: Furuta_U assay: sensitivity 66.7%, specificity 100%, cut-off943.1, Area under the curve (AUC) is 0.939. The 95% CI range was 0.817to 0.989 at a significance of P=0.0001 for area=5.

FIG. 6 d: Qiu_U assay: sensitivity 83.3%, specificity 94.1%, cut-off431.1, Area under the curve (AUC) is 0.944. The 95% CI range was 0.824to 0.990 at a significance of P=0.0001 for area=5.

FIG. 6 e: Summary table of results obtained for each of the four assays.

FIG. 7: MAGE-A3 methylation status in lung biopsies: Receiver OperatingCharacteristics (ROC) curves were calculated for the 4 MAGE-A3Unmethylated assays by plotting the true positive rate (sensitivity) infunction of the false positive rate (100-specificity).

FIG. 7 a: GO_(—)1_U assay: sensitivity 84.6%, specificity 91.7%, cut-off115.8, Area under the curve (AUC) is 0.954. The 95% CI range was 0.868to 0.990 at a significance of P=0.0001 for area=5.

FIG. 7 b: GO_(—)2_U assay: sensitivity 88.5%, specificity 94.4%, cut-off108.28, Area under the curve (AUC) is 0.971. The 95% CI range was 0.893to 0.996 at a significance of P=0.0001 for area=5.

FIG. 7 c: Furuta_U assay: sensitivity 84.6%, specificity 91.7%, cut-off296.8, Area under the curve (AUC) is 0.949. The 95% CI range was 0.861to 0.988 at a significance of P=0.0001 for area=5.

FIG. 7 d: Qiu_U assay: sensitivity 84.6%, specificity 91.7%, cut-off176.71, Area under the curve (AUC) is 0.948. The 95% CI range was 0.859to 0.988 at a significance of P=0.0001 for area=5.

FIG. 7 e: Summary table of results obtained for each of the four assayson lung biopsies.

FIG. 8: MAGE-A3 methylation status in lung FFPE samples: ReceiverOperating Characteristics (ROC) curves were calculated for the 4 MAGE-A3Unmethylated assays by plotting the true positive rate (sensitivity) infunction of the false positive rate (100-specificity).

FIG. 8 a: GO_(—)1_U assay: sensitivity 84.0%, specificity 96.0%, cut-off21.88, Area under the curve (AUC) is 0.933. The 95% CI range was 0.825to 0.984 at a significance of P=0.0001 for area=5.

FIG. 8 b: GO_(—)2_U assay: sensitivity 84.0%, specificity 96.3%, cut-off17.75, Area under the curve (AUC) is 0.932. The 95% CI range was 0.826to 0.983 at a significance of P=0.0001 for area=5.

FIG. 8 c: Furuta_U assay: sensitivity 80.0%, specificity 96.2%, cut-off214.26, Area under the curve (AUC) is 0.923. The 95% CI range was 0.813to 0.979 at a significance of P=0.0001 for area=5.

FIG. 8 d: Qiu_U assay: sensitivity 72.0%, specificity 96.2%, cut-off68.91, Area under the curve (AUC) is 0.912. The 95% CI range was 0.799to 0.973 at a significance of P=0.0001 for area=5.

FIG. 8 e: Summary table of results obtained for each of the tour assayson lung FFPE samples.

FIG. 9: Effect of melanin on PCR inhibition when spiked at differentsteps of the reaction process

FIG. 9 a: LNCaP cell line material with and without spiked melaninprocessed through MAGE-A3 U real-time MSP. BT=bisulphite treatment.

FIG. 9 b: MCF7 cell line material with and without spiked melaninprocessed through Gst-Pi M real-time MSP

FIG. 10: Protein D-MAGE-A3-His

-   SINGLE UNDERLINED=first 109 amino acids of Protein D-   DOUBLE UNDERLINED=Protein D signal sequence (18 aa)-   =inserted/substituted sequences: Met-Asp at 2-3 (substituted);    Met-Asp at 128-129(inserted) and Gly-Gly at 442-443 (inserted)-   Bold=fragment of MAGE3: amino acids 3-314 of MAGE3 (312 AA total)-   Grey=7 his tail

DETAILED DESCRIPTION—EXPERIMENTAL SECTION Example 1 Real Time AmplifluorAssay

A direct real-time fluorescence based methylation-specific PCR assay(real-time MSP assay) was developed to define the methylation status ofthe MGMT promoter (Vlassenbroeck et al., J Mol Diagn 2008, 10:332-337).This technology is illustrated and summarised in the figure legend forFIG. 4 on page 70.

Analyte quantitations for Mage-A3 were successfully performed using thistechnology. This consisted of parallel amplification/quantificationprocesses using specific primer and primer/detector pairs for Mage-A3using the Amplifluor® assay format on an ABI Prism® 7900HT instrument(Applied Biosystems).

The final primer concentrations in the reaction mix were 100 nM for bothforward primer/detector and reverse primer. 12.5 μl of iTaq™ Supermixwith Rox (BioRad, 2×buffer) were used per PCR reaction. The total volumeper reaction, including 5 μl of modified template DNA, was 25 μl. TheABI 7900HT SDS instrument was started 10 min before use, allowing theheated cover to reach 105° C. The following thermal profile was used:Stage1: 50° C. for 2 min, Stage2; 95° C. for 10 min, Stage3: 95° C. for15 sec, 62° C. for 1 min (=plateau-data collection) for 45 repeats.

Plasmid material, used as standard curve was generated as follows: thepromoter sequence as defined by the primers is PCR amplified and cloned(using suitable isolated and bisulphite modified cell line DNA). Thesequence is verified by sequencing and compared to the publishedpromoter sequence.

A standard curve (2×10⁶—20 copies) was included to determine copynumbers of unknown samples by interpolation of their Ct values to thestandard curve. B-Actin was used as a reference gene in the assay.

Example 2 MAGE-A3 Assays and Primers Design

Primers useful for detecting unmethylated MAGE-A3 as described in Qiu etal.: Clinical Biochemistry 39 (2006), 259-2; Jang et al.: CancerResearch 61 (2001), 7959-7963 and Furuta et al.: Cancer Sci 95 (2004),962-968 were synthesised, and are shown in Table 1 in addition to novelprimer sequences.

In silico design of forward (F) and reverse (R) primers for detectingunmethylated or alternatively methylated form of Mage A3 were done usingPrimer3 software adapted to MSP requirements(http://fokker.wi.mit.edu/primer3/input.htm). Conditions were asfollows: amplicon size: 60-120 nt; primer size: 18-27 nt; melting temp:55-65° C.; max 3′ self complementarity=0; Window of 200 bp around TSS(number to return=2000) .

The U_primers were designed for detecting unmethylated Mage-A3 whereasthe M_primers were designed for detecting methylated Mage-A3. Finally,primers A MAGE_A3 and MAGEA3_GO_(—)1_U_F, MAGEA3_GO_(—)2_U_R,MAGEA3_GO_(—)1_U_R_AMP, MAGEA3_GO_(—)2_U_F_AMP, MAGEA3_GO_(—)1_M_F,MAGEA3_GO_(—)2_M_F, MAGEA3_GO_(—)1_M_4_AMP and MAGEA3_GO_(—)2_M_R_AMPwere retained for further investigation. Location of the U-primersrelative to the Transcription Start Site (TSS) is shown in FIG. 1 andFIG. 2. The primers are positioned around the Transcription Start Site.

Either the forward or reverse primer was synthesised to incorporate asuitable stem loop or hairpin structure carrying a donor and acceptormoiety at the 5′ end having the nucleotide sequence: 5′AGCGATGCGTTCGAGCATCGCU 3′ (SEQ ID NO 1.)

Different MAGEA3 primer combinations were tested. Finally 4 U-assays and2 M-assays were retained for further development. The selected primercombinations for each assay are summarized in Table 1.

TABLE 1 Primer and amplifluor detector sequences MAGEA3 Assay- 5′ to 3′Sequences Amplicon Detector Modifications: 5′ FAM Name lengthand internal dUdabcyl MAGEA3_GO_1_U_F U assay ATTTTTGTTTGGAATTTAGGGTAGForward primer (set 2)- (SEQ ID NO. 2) MAGEA3_GO_1_U_R_AMP 142 bpAGCGATGCGTTCGAGCATCGCUCCAACATCAAACC Reverse detectorATCACTCA (SEQ ID NO. 3) MAGEA3_GO_2_U_F_AMP U assayAGCGATGCGTTCGAGCATCGCUTGGAATTTAGGGT Forward detector (set 3)-AGTATTGT (SEQ ID NO. 6) MAGEA3_GO_2_U_R 140 bp CCCTCCACCAACATCAAAReverse primer (SEQ ID NO. 7) MAGEA3_FURUTA_U_F_AMP U assayAGCGATGCGTTCGAGCATCGCUTTAGGATGTGATG Forward detector (set 7)-TTATTGATTTGT (SEQ ID NO. 9) MAGEA3_FURUTA_U_R 110 bpCCAACATCAAACCATCACTCA Reverse primer (SEQ ID NO. 4) MAGEA3_QIU_U_F_AMPU assay AGCGATGCGTTCGAGCATCGUTGTTTGGAATTTA Forward detector (set 9)-GGGTAGTATTGT (SEQ ID NO. 12) MAGEA3_QIU_U_R 126 bpCCATCACTCATTACTCAAAACAAA Reverse primer (SEQ ID NO. 13) ACTB_F_AMPReference- AGCGATGCGTTCGAGCATCGCUTAGGGAGTATATA Forward detector 125 bpGGTTGGGGAAGTT (SEQ ID NO. 21, or SEQ ID NO: 1 + SEQ ID NO: 20) ACTB_RAACACACAATAACAAACACAAATTCAC Reverse primer (SEQ ID NO. 22)MAGEA3_GO_1_M_F M assay ATTTTTGTTCGGAATTTAGGGTAG Forward primer (set 2)-(SEQ ID NO. 14) MAGEA3_GO_1_M_R_AMP 142 bpAGCGATGCGTTCGAGCATCGCUCCGACGTCAAACC Reverse detectorGTCGCTCG (SEQ ID NO. 15) MAGEA3_GO_2_M_F M assay CGGAATTTAGGGTAGTATCGTForward primer (set 4)- (SEQ ID NO. 17) MAGEA3_GO_2_M_R_AMP 140 bpAGCGATGCGTTCGAGCATCGCUCCCTCCGCCGACG Reverse detectorTCAAA (SEQ ID NO. 18)

Example 3 Analytical Assay Performance

The analytical performance (detection limit and specificity) of theassay was demonstrated using reconstructed substrates.

Limit of Detection

To determine the sensitivity of MSP for the unmethylated pattern,positive confirmed cell line material (LNCaP and Gerl), was seriallydiluted and mixed with control (negative) cell line DNA (DU145).Dilutions of 1/10; 1/100and 1/500 were made (see Table 2). A totalamount of 750 ng of DNA (U DNA+M DNA) was bisulphite treated using theEZ DNA methylation kit from Zymo Research.

TABLE 2 Dilution scheme cell mixtures U DNA (ng) M DNA (ng) [LNCaP orGerl] [DU145] 750 ng 0 ng 75 ng 675 ng 7.5 ng 742.5 ng 1.5 ng 748.5 ng 0ng 750 ng

Subsequently 2.4 μl of the chemically treated DNA was used as templatefor MAGEA3 real-time MSP using specific primers for the unmethylatedGO_(—)2_U assay (LNCaP/DU145 DNA mixture) and unmethylated Furuta assay(Gerl/DU145 DNA mixture). Results are presented in FIG. 3.

As can be seen, the lower detection limit of the MAGEA3 GO_(—)2_U andMAGEA3_Furuta real-time MSP was repeatedly set at 1.5 ng (1/500dilution), this considering the whole sample preparation procedure.Since 10% of the sample is used per PCR reaction the final analyticalsensitivity is 0.15 ng.

Analytical Specificity

The specificity of the MAGEA3 GO_(—)1_U/GO_(—)2_U/Furuta_U and QIU_Uprimer set was confirmed by MSP using CpGenome™ UniversalMethylated/Unmethylated DNA (Chemicon International, CA, USA; Cat. #S7821 and Cat. # S7822) and subsequent agarose gel analysis. Briefly,amplifluor real-time MSP was performed on the I Cycler (Bio-Rad) usingthe following thermal profile: Stage1: 50° C. for 2 min, Stage2: 35° C.for 10 min, Stage3: 95° C. for 15 sec, 62° C. for 1 min (=plateau-datacollection) for 45 repeats. As a high specificity is essential forAmplifluor-based detection, a temperature gradient was applied in stage3 to select for the best annealing temperature (57° C., 58.1° C., 60.3°C. and 61.8° C.).

All resulting PCR products were run on a 3% agarose gel. No band wasvisualized when CpGenome™ Universal Methylated DNA was used as templateDNA (tested at 57° C.), confirming specificity for the Unmethylated DNA.

In addition, the specificity of the MAGEA3 assays was investigatedamongst other gene members of the MAGE-A family using sequencealignment. The number of mismatches of the MAGE-A3GO_(—)1_U/GO_(—)2_U/Furuta_U and QIU_U primerset vs. MAGE-A2 andMAGE-A12 sequences (converted sequences) is indicated in Table 3. Theinvestigated U primers appeared specific for MAGE-A3 U/MAGE-A6 U.

TABLE 3 Sequence alignment MAGE-A2 MAGE-A12 Assays: Primers: MismatchMismatch GO_1 U Forward 3 3 Reverse 1 1 Total 4 4 GO_2 U Forward 6 6Reverse 0 0 Total 6 6 Furuta U Forward 1 2 Reverse 1 1 Total 2 3 Qiu UForward 6 6 Reverse 6 6 Total 12 12

Cloning MAGE-A3 Regulatory Sequences and Performance Standard Curve

A regulatory MAGE-A3 U DNA sequence of 364 bp was cloned using theflanking primers as indicated in Table 4.

TABLE 4 Flanking primers used to generate MAGEA3 plasmid material TargetSense (S), Flanking or gene Antisense Sequence primers name (A) 5′ to 3′MAGEA3_FL_1_S MAGEA3 S ATTTTGAGGGATGATC GAAG (SEQ ID NO 23)MAGEA3_FL_1_AS MAGEA3 A CTAAAATAAAACCCGC CTCA (SEQ ID NO 24)

This cloned material was used as standard curve material for Real TimeMSP. The reproducibility was first confirmed by running 2 plates of 6standard curves (2*10⁶-2*10¹ copies) (2 different operators, 3 PCRmixes/operator/plate). Slope, PCR efficiency and R² values weremonitored and gave acceptable results.

Performance Standard Curve.

A serial dilution of MAGEA3 plasmid material (2×10⁶ to 2×10¹ copies/5μl) was loaded in duplicate using the specified primer and Amplifluordetector sequence in Table 1 with following optimized thermal profile:Stage1: 50° C. for 2 min, Stage2: 95° C. for 10 min, Stage3: 95° C. for15 sec, 59° C. for 30 sec, 59° C. for 30 sec (=plateau-data collection)for 45 repeats. Results were generated using the SDS 2.2 software(Applied Biosystems), exported as Ct values (cycle number at which theamplification curves cross the threshold value, set automatically by thesoftware). The performance of the standard curve is shown in Table 5

TABLE 5 Summary of slopes and PCR efficiencies MAGEA3 Name Slope R²Efficiency MAGEA3_U set2 standard 3.6736 0.9999 87.2% curve (plasmid)MAGEA3_U set3 standard 3.6994 0.9998 86.3% curve (plasmid) MAGEA3_U set7standard 3.6108 0.9994 89.2% curve (plasmid) MAGEA3_U set9 standard3.4885 0.9988 93.5% curve (plasmid) MAGEA3_M set2 standard 3.8601 0.999781.6% curve (plasmid) MAGEA3_M set4 standard 3.6701 0.9997 87.3% curve(plasmid)

Example 4 Performance of the Assay on Cell Line Material

The MAGEA3 methylation status was investigated for 19 cell lines. Thebest methylated and unmethylated cell lines for the MAGE A3 U and Massay respectively are displayed below.

TABLE 6 Cell lines processed through MAGEA3 U assays Cell β-ActinPrimers set U_2 Primers set U_3 Primers set U_7 lines: Ct: copies Ct:copies ratios Ct: copies ratios Ct: copies ratios Gerl 29.28 2086 29.571403 673 29.73 1753 841 26.97 2431 1160 (108216) Staq 28.09 4484 35.9326 6 UND 34.41 16 4 (108217) CRL9609 27.58 6192 32.47 225 36 34.98 73 1229.80 358 58 (108218) LNCaP 28.82 2814 28.54 2684 954 28.64 3364 119525.88 5085 1807 Du145 29.07 2393 >40 UND >40 HL60 28.29 3940 39.89 2.100.53 >40 >40

TABLE 7 Cell lines processed through MAGEA3 M assays β-Actin Primers setM_2 Primers set M_4 Cell lines: Ct: copies: Ct: copies: ratios: Ct:copies: ratios: Gerl (108216) 29.28 2086 >40 >40 Staq (108217) 28.094484 29.09 1388 309 27.54 2100 468 CRL9609 (108218) 27.58 6192 27.633363 543 26.89 3200 517 LNCaP 28.82 2814 >40 >40 Du145 29.07 2393 29.72945 395 28.96 839 351 HL60 28.29 3940 28.97 1492 379 27.91 1657 421

Example 5 Intermediate Precision

The intermediate precision was tested by repeatedly performing the sameassay for the unmethylated and methylated version of the MAGEA3 promotersequence. Different numbers of fully modified MAGEA3 U and MAGEA3 Mpromoter DNA molecules (standard curve material) were measuredrepeatedly. In addition the operator factor was tested by having 2different skilled laboratory people (operators A and B) perform theassay repeatedly on 2 different days (3 different standard curvedilutions in duplicate were run per operator per day). Table 8 and 9summarize experiments done to test the intermediate precision of theGO_(—)1 and GO_(—)2 and M MAGEA3 assay. It was shown that the standarddeviations of all results referring to the same numbers of moleculesrange between 0.11 and 1.29. A summary of all correlation coefficients(different operators and days) are shown in Table 9, average R² rangebetween 0.9959 and 0.9997).

TABLE 8 Assays performed to test the intermediate precision (operator Aand B on 2 different days): column 1: numbers of molecules (log)following colums: mean Ct values and standard deviation for each MAGEA3assay GO_1_U GO_2_U Furuta GO_1_M GO_2_M Average of Average of Averageof Average of Average of Log all Ct all Ct all Ct all CT all CT copiesvalues (SD) values (SD) values (SD) values (SD) values (SD) 6.30 17.69(0.27) 19.31 (0.33) 17.23 (0.84) 17.78 (0.47) 17.73 (0.12) 5.30 21.41(0.23) 22.98 (0.37) 20.70 (0.87) 21.55 (0.44) 21.32 (0.11) 4.30 25.00(0.27) 26.64 (0.39) 24.30 (0.86) 25.36 (0.46) 24.90 (0.11) 3.30 28.70(0.37) 30.34 (0.68) 27.85 (0.78) 29.21 (0.36) 28.51 (0.14) 2.30 32.77(1.28) 34.06 (0.46) 31.42 (0.85) 33.26 (0.52) 32.10 (0.38) 1.30 36.24(1.16) 37.86 (1.29) 34.97 (1.20) 37.23 (1.20) 35.75 (1.25) R² = 0.9997R² = 1.000 R² = 1.000 R² = 0.9998 R² = 1.000

TABLE 9 Correlation coefficients found for each analyzed dilutionseries. oper- R² R² R² R² R² day ator points GO_1_U GO_2_U Furuta GO_1_MGO_2_M 1 A 6 0.9993 0.9974 0.9999 0.9972 0.9996 1 A 6 0.9993 0.99980.9993 0.9991 0.9992 1 A 6 0.9997 0.9969 0.9992 0.9988 0.9994 1 B 60.9997 0.9989 0.0999 0.9996 0.9999 1 B 6 0.9996 0.9971 0.9998 0.99860.9996 1 B 6 0.9983 0.9987 0.9999 0.9998 0.9959 2 A 6 0.9950 0.99960.9997 0.9989 0.9912 2 A 6 0.9988 0.9966 0.9998 0.9990 0.9994 2 A 60.9997 0.9957 0.9996 0.9970 0.9997 2 B 6 0.9995 0.9994 0.9999 0.99760.9980 2 B 6 0.9975 0.9997 0.9998 0.9998 0.9999 2 B 6 0.9648 1.00000.9992 0.9976 0.9999 Aver- Aver- Aver- Aver- Aver- age age age age age0.9959 0.9983 0.9997 0.9986 0.9985

Example 6 Melanin Interference

Previously it has been reported that the efficiency of PCRs from samplescontaining melanin was low.

Eckhart et al. (2000) found that both RNA and cDNA preparations derivedfrom melanocytes contain a RT-PCR inhibitor that copurified with nucleicacids. Investigation of the candidate inhibitor melanin revealed that itreversibly binds to thermostable DNA polymerase and inhibits itsactivity. Before processing melanoma samples through the MAGEA3 Uamplifluor assays, the potential inhibition by melanin was investigated.

Synthetic melanin was prepared as described by Eckhart et al. Briefly,melanin (SIGMA M8631) was dissolved in distilled water at aconcentration of 2 mg/ml, vortexed extensively and sonicated in a waterbath at room temperature for 10 min. The non-dissolved melanin wasremoved by centrifugation at 9000 g. The potential inhibition effect wastested by adding melanin at different steps of the reaction process:

-   1) Before extraction: 1 μg and 5 μg of prepared melanin was added to    250,000 LNCaP cells and 250,000 MCF7 cells-   2) After extraction: 1 μg and 5 μg of prepared melanin was added to    1 μg of LNCaP and MCF7 DNA-   3) After bisuphite treatment: 1 μg and 5 μg of prepared melanin was    directly spiked in the PCR reaction. Bisulphite elution volumes were    adapted to have either a constant template concentration (annotated    as ‘a’, e.g. LNCaP a) or a constant amount of template in the PCR    reaction (annotated as ‘b’, e.g. LNCaP b).

These melanin containing LNCaP and MCF7 samples were simultaneouslyprocessed with corresponding non-spiked melanin samples.

Samples were processed further using PUREGENE® DNA Purification Kit andEZ DNA Methylation kit. The chemically modified DNA was used as inputmaterial tor MAGEA3 U, Gst-Pi H and ACTB real-time MSP.

Recovered copy numbers of the tested gene promoter and ACTB referencegene were calculated and compared for each condition.

Results are shown in FIG. 9. No significant PCR inhibitory effect wasobserved when melanin was added before or after DNA extraction. Melaninonly showed clear inhibition when directly spiked into the PCR reaction.Contrary to RT-PCR, melanoma samples with high melanin content can beprocessed through real-time MSP without risk of PCR inhibition.

Example 7 MAGE -A3 Methylation Status in Melanoma/Lung Samples andConcordance with RNA Expression Materials and Methods Clinical Samples

Surgical specimens from melanoma and lung cancer patients were providedby GSKBio: genomic DNA samples (gDNA), biopsy material in RNA later®solution and corresponding formalin fixed paraffin embedded tissue(FFPE) were classified as MAGEA3 positive or MAGEA3 negative based onGSKBio RNA expression data. An overview of the provided sample set isdetailed in Table 10.

TABLE 10 Clinical sample collection MAGEA3 RNA expression DiagnosisSample Number of classification by group type samples RT-PCR MelanomasqDNA 41 24 positive 17 negative NSCLC tissue in 61 26 positive RNAlater ® 35 negative NSCLC FFPE 52  26 positive*  26 negative**classification was made based on the corresponding RNA later  ® tissue

Cell Lines:

Cell lines were included in each run as positive and negative controls.Before applying the amplifluor real-time MSP assay on clinical samples,the sensitivity and specificity of the assay was affirmed on cell linematerial. The best MAGEA3 methylated and unmethylated cell lines aresummarized in Table 11. Gerl, Staq en CRL9609 were obtained from GSKBio,cell lines LNCaP and DU145 were purchased from the American Type CultureCollection.

MAGEA3 RNA MAGEA3 expression methylation status status Gerl Positiveunmethylated Staq Negative methylated CRL9609 Negative methylated LNCaPNot tested unmethylated DU145 Not tested methylated

DNA Isolation:

Formalin Fixed paraffin embedded samples were first de-paraffinized in750 μl xylene for 2 h. A second xylene treatment was done (400 μl xylenefor 2 h) Then, 250 μl of 70% ethanol was added before centrifagation at13000 rpm for 15 min. The supernatant was removed and the samples wereair dried at room temperature.

The samples in RNA later® were cut in very small pieces using- a razorblade after removal of the RNA later® solution.

Subsequently, the DNA was extracted using the classicalphenol/chloroform extraction method and resuspended in 50 μl LoTE (3 mMTRIS, 0.2 mM EDTA, pH 8.0).

DNA was quantified using the Picogreen® dsDNA quantitation kit(Molecular Probes, #P7589) following the manufacturer's recommendations.λDNA provided with the kit was used to prepare a standard curve. Thedata were collected using a FluoStar Galaxy plate reader (BMG Labtechnologies, Germany).

DNA modification: 1.5 μg of DNA was subjected to bisulphite modificationusing the EZ DNA Methylation kit from Zymo Research.

Briefly, aliquots of 45 μl were mixed with 5 μl of M-Dilution Buffer andincubated at 37° C. for 15 min shaking at 1100 rpm. Then 100 μl of thediluted CT Conversion Reagent was added and samples were incubated at70° C. for 3 h, shaking at 1100 rpm in the dark. After conversion, thesamples were further desalted and desulfonated according tomanufacturer's instructions and eluted in 25 μl Tris-HCl 1 mM pH8.0. Themodified DNA was stored at −80° C. until further processing.

DNA amplification: Real-time MSP was applied on a 7900HT fast real-timePCR cycler from Applied Biosystems.

Four MAGEA3 hypomethylation assays, designed to target the unmethylatedversion of the gene promoter sequence were tested for concordance withthe provided RNA expression data, which was measured in accordance withmethods described in WO2007/47876, for example, using the primers andprobe of Table 2, Exon 3 MAGE-A3 specific primers and probe of SEQ IDNO:3, 4 and 13. The independent reference gene β-actin (ACTB) was alsomeasured. Primer and amplifluor detector sequences are shown in Table 1.

2.4 μl of the modified genomic DNA sample was added to a final 12 μl PCRreaction volume including: 6 μl of iTaq™ Supermix with Rox (BioRad,2×buffer) and final primer concentrations of 100 nM for both forwardprimer/detector and reverse primer. Cycling conditions for each MAGEA3design were 50° C. for 2 min; 95° C. for 10 min; followed by 45 cyclesof 95° C. for 15 sec, 59° C. for 30 sec [62° C. for ACTB] and 59° C. for30 sec [62° C. for ACTB] (=plateau-data collection).

Results were generated using the SDS 2.2.2 software (AppliedBiosystems), exported as Ct values (cycle number at which theamplification curves cross the threshold value, set automatically by thesoftware), and then used to calculate copy numbers based on a linearregression of the values plotted on a standard curve of 20-2×10̂6 genecopy equivalents, using plasmid DNA or purified PCR products containingthe bisulphite modified sequence of interest. The ratio between MAGEA3and ACTB was calculated to generate the test result. In order tointerpret the data, a clinical cutoff (threshold) was defined based onthe un-blinded RNA expression data.

The samples were classified as methylated, non-methylated, or invalidbased on the decision tree shown in FIG. 5. Cell lines were included ineach run as positive and negative controls, and entered the procedure atthe DNA extraction step.

A run was considered valid when the following criteria were met: a) PCRefficiency of both standard curves above 80%; b) r̂2 of at least 4relevant data points above 0.990; c) Δ Ct between duplicates <1.5; d)routinely included NTC not amplified; e) 10% of a 1.5 μg conversionreaction of the positive cell line assay control was detectable; and f)10% of a 1.5 μg conversion reaction of the negative cell line assaycontrol was not detected within the standard curve.

Results

Concordance between Methylation and Gene Expression:

Melanomas:

Expression and methylation levels of MAGEA3 were compared on a samesample set. In total, 41 melanoma samples were processed using RT-PCRand real-time MSP. Several designs of the MAGEA3 U amplifluor assay weretested to see which assay accorded best with the RNA expression dataprovided by GSK. The clinical cut-off was set in such a way havingmaximum concordance and minimum of false positives (see Table 12). ROCcurves for the MAGEA3 methylation status in these samples are shown inFIG. 6. Among the 17 negative samples, 9 were positive for other MAGE-Afamily members, the GO_(—)2_U and Furuta U assay correctly classifiedthese 9 samples (specificity of 100%).

Taken all this data together, the MAGEA3_GO_(—)2_U assay performed bestwith a 92.7% concordance and 100% specificity.

TABLE 12 Concordance data melanoma samples MAGEA3_ MAGEA3_ MAGEA3 MAGEA3GO_1_U GO_2_U Furuta U Qiu U Cut off 315 330 946 434 Correctlyclassified 13 17 17 16 for MAGEA3 negatives (GSK → 17) Correctlyclassified 22 21 16 20 for MAGEA3 positives (GSK → 24) Correctlyclassified 35 38 33 36 samples (total samples: 41) Concordance 85.4%92.7% 80.5% 87.8%

Lung Samples (Biopsies and FFPE):

The same set up as above was tested on a different sample set: 52 lungFFPE samples and 61 lung tissues in RNA later® were screened through the4 MAGEA3 U amplifluor assays and accorded with corresponding RNA data.

ROC curves for the MAGEA3 methylation status in these lung biopsy andFFPE samples are presented in FIGS. 7 and 8 respectively. Among theMAGEA3 negative samples, 9 were positive for other MAGE-A familymembers; the MAGEA3 U assays correctly classified all 9 samples(specificity of 100%). Obtained results confirmed that theMAGEA3_GO_(—)2_U assay was the best performing assay with a concordanceof 90.4% in FFPE and a concordance of 91.8% in biopsies, maintaining aspecificity of 100% (Table 13).

TABLE 13 Concordance data lung samples (MAGEA3_GO_2_U assay)MAGEA3_GO_2_U FFPE Biopsies Cut off 29 112 Correctly classified for26/26 33/35 MAGEA3 negatives (GSK) Correctly classified for 21/26 23/26MAGEA3 positives (GSK) Correctly classified 47 56 Total samples 52 61Concordance 90.4% 91.8%

Example 8 Testing of Lung Samples through MAGEA3 Assays

DNA from lung cancers was processed through MAGEA3 (U & M assayversions) and β-actin assays in parallel with LNCaP & DU145 control celllines. Several designs of MAGEA3 U amplifluor assay were tested to seewhich assay accorded best with the MAGEA3 GO_(—)2 U assay. Experimentalconditions as described in example 7 were used. Standard curves showedefficiencies above 80%. R2 was higher than 0.99. Cut off values were setat 29 for for the MAGEA3 GO_(—)2 U assay; 22 for the MAGEA3 GO_(—)1assay; 229 tor the MAGEA3 Furuta U assay; 87 for the MAGEA3 Qiu assay;148 or the MAGEA3 GO_(—)1 M assay and 167 for the MAGEA3 GO_(—)2 Massay. Methylation levels of MAGEA3 were compared on the same sampleset. Results are shown in Tables 14 and 15. For the MAGEA3 GO_(—)2 Uassay (cut off=29):

-   -   3 samples are classified as invalid and those samples are not        included for the comparison with the others U & M assays;    -   3 samples are classified as non-methylated;    -   15 samples are classified as methylated.

The four MAGEA3 U assays give similar results with 96% of concordancewith the MAGEA3 GO_(—)2 U assay for the valid samples. The MAGEA3 Massays gave 71% and 75% concordance with the MAGEA3 GO_(—)2 U assay.

TABLE 14 Summary table comparing the different U assays and showing theconcordances calculated for the U assays compared to GO_2 U assay (thistable takes only the valid samples into account). MAGEA3 MAGEA3 MAGEA3 Uset MAGEA3 U set Uset 7 (Furuta U, U set 3 (GO 2 U) 2 (GO 1 U) REPEAT) 9(Qiu U) METHYLATED 15 14 14 14 (/15): NON- 9 9 9 9 METHYLATED (/9):TOTAL 24 23 23 23 Concordance: 100% 96% 96% 96%

TABLE 15 Summary table comparing the different M assays and showing theconcordances calculated for the M assays compared toGO_2 U assay (thistable takes only the valid samples into account). MAGEA3 MAGEA3 MAGEA3 Uset 3 M set 2 M set 4 (GO 2 U) (GO 1 M) (GO 2M) METHYLATED 15 11 13(/15): NON- 9 6 5 METHYLATED (/9): TOTAL 24 17 18 Concordance: 100% 71%75%

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention In addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. Moreover, all embodiments described herein areconsidered to be broadly applicable and combinable with any and allother consistent embodiments, as appropriate.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An oligonucleotide comprising a primer or a probe, a primer or aprobe comprising or consisting essentially of or consisting of thenucleotide sequence of any of SEQ ID NO. 5, 6, 7, 2, 3, 4, 8, 9, 11, 12,13, 14, 15, 16, 17, 18, 19 or 25 which oligonucleotide, primer or probeis useful for the detection of the methylation status of a gene.
 2. Anoligonucleotide, primer or probe according to claim 1 comprising,consisting essentially of or consisting of the nucleotide sequence ofany of SEQ ID NO. 5, 6, 7, 2, 3, 4 or 25 which oligonucleotide, primeror probe is useful for the detection of the methylation status of agene.
 3. An oligonucleotide, primer or probe according to claim 1comprising or consisting essentially of or consisting of the followingcontiguous sequences in 5′ to 3′ order. (a) a first nucleotide sequenceof between approximately 6 and 30 nucleotides, wherein a nucleotidewithin said first nucleotide sequence is labelled with a first moietyselected from the donor moiety and the acceptor moiety of a molecularenergy transfer pair, wherein the donor moiety emits fluorescence at oneor more particular wavelengths when excited, and the acceptor moietyabsorbs and/or quenches said fluorescence emitted by said donor moiety;(b) a second, single-stranded nucleotide sequence comprising, consistingessentially of or consisting of between approximately 3 and 20nucleotides; (c) a third nucleotide sequence comprising, consistingessentially of or consisting of between approximately 6 and 30nucleotides, wherein a nucleotide within said third nucleotide sequenceis labelled with a second moiety selected from said donor moiety andsaid acceptor moiety, and said second moiety is the member of said groupnot labelling said first nucleotide sequence, wherein said thirdnucleotide sequence is complementary in reverse order to said firstnucleotide sequence such that a duplex can form between said firstnucleotide sequence and said third nucleotide sequence such that saidfirst moiety and second moiety are in proximity such that, when thedonor moiety is excited and emits fluorescence, the acceptor moietyabsorbs and quenches said fluorescence emitted by said donor moiety; and(d) at the 3′ end of the primer, a fourth, single-stranded nucleotidesequence comprising, consisting essentially of or consisting of betweenapproximately 8 and 40 nucleotides that comprises or consistsessentially of or consists of at its 3′ end a sequence of any of SEQ IDNO. 5, 7, 2, 4, 8, 11, 13 or 25 (and able to prime synthesis by anucleic acid polymerase of a nucleotide sequence complementary to anucleic acid strand comprising the portion of the unmethylated DNA ofthe MAGE A3 gene); wherein, when said duplex is not formed, said firstmoiety and said second moiety are separated by a distance that preventsmolecular energy transfer between said first and second moiety.
 4. Anoligonucleotide, primer or probe according to claim 1 comprising orconsisting essentially of or consisting of the following contiguoussequences in 5′ to 3′ order. (a) a first nucleotide sequence of betweenapproximately 6 and 30 nucleotides, wherein a nucleotide within saidfirst nucleotide sequence is labelled with a first moiety selected fromthe donor moiety and the acceptor moiety of a molecular energy transferpair, wherein the donor moiety emits fluorescence at one or moreparticular wavelengths when excited, and the acceptor moiety absorbsand/or quenches said fluorescence emitted by said donor moiety; (b) asecond, single-stranded nucleotide sequence comprising, consistingessentially of or consisting of between approximately 3 and 20nucleotides; (c) a third nucleotide sequence comprising, consistingessentially of or consisting of between approximately 6 and 30nucleotides, wherein a nucleotide within said third nucleotide sequenceis labelled with a second moiety selected, from said donor moiety andsaid acceptor moiety, and said second moiety is the member of said,group not labelling said first nucleotide sequence, wherein said thirdnucleotide sequence is complementary in reverse order to said firstnucleotide sequence such that a duplex can form between said firstnucleotide sequence and said third nucleotide sequence such that saidfirst moiety and second moiety are in proximity such that, when thedonor moiety is excited and emits fluorescence, the acceptor moietyabsorbs and quenches said fluorescence emitted by said donor moiety; and(d) at the 3′ end of the primer, a fourth, single-stranded nucleotidesequence comprising, consisting essentially of or consisting of betweenapproximately 8 and 40 nucleotides that comprises or consistsessentially of or consists of at its 3′ end a sequence of any of SEQ IDNO. 14, 16, 17 or 19 (and able to prime synthesis by a nucleic acidpolymerase of a nucleotide sequence complementary to a nucleic acidstrand comprising the portion of the methylated DNA of the MAGE A3gene); wherein when said duplex is not formed, said first moiety andsaid second moiety are separated by a distance that prevents molecularenergy transfer between said first and second moiety.
 5. Anoligonucleotide, primer or probe according to claim 3 wherein thefourth, single-stranded nucleotide sequence comprises, consistsessentially of or consists of between approximately 8 and 40 nucleotidesthat comprises at its 3′ end a sequence of any SEQ ID NO. 5, 7, 2, 4 or25.
 6. Primer pair comprising a primer according to claim
 1. 7. Primerpair comprising a primer according to claim
 5. 8. Primer pair comprisingor consisting essentially of or consisting of the nucleotide sequence ofSEQ ID NO. 6 and 7; SEQ ID NO. 2 and 3; SEQ ID NO. 9 and 4; SEQ ID NO.12 and 13; SEQ ID NO. 14 and 15; or SEQ ID NO. 17 and
 18. 9. Primer pairaccording to claim 8 comprising or consisting essentially of orconsisting of the nucleotide sequence of SEQ ID NO. 6 and 7 or SEQ IDNO. 2and
 3. 10. A kit for detecting the methylation status of a genecomprising at least one oligonucleotide, primer or probe as defined inclaim
 1. 11. An oligonucleotide, primer or probe as defined in claim 1,wherein said gene is the MAGE-A3 gene.
 12. Method of detecting thepresence and/or amount of unmethylated Mage-A3 gene in a DNA-containingsample, comprising: (a) contacting/treating the DNA-containing samplewith a reagent which selectively modifies unmethylated cytosine residuesin the DNA to produce detectable modified residues but which does notmodify methylated cytosine residues (b) amplifying at least a portion ofthe unmethylated gene of interest using at least one primer pair, atleast one primer of which is designed to bind only to the sequence ofunmethylated DNA following treatment with the reagent, wherein at leastone primer in the primer pair comprises, consists essentially of, orconsists of the nucleotide sequence of any of SEQ ID NO. 5, 6,7, 2, 3,4, 8, 9, 11,12, 13 or
 25. 13. Method according to claim 12 wherein atleast one primer in the primer pair comprises, consists essentially of,or consists of the nucleotide sequence of any of SEQ ID NO. 5, 6, 7, 2,3, 4 or
 25. 14. Method of detecting the presence and/or amount ofmethylated Mage-A3 gene in a DNA-containing sample, comprising: (a)contacting/treating the DNA-containing sample with a reagent whichselectively modifies unmethylated cytosine residues in the DNA toproduce detectable modified residues but which does not modifymethylated cytosine residues (b) amplifying at least a portion of themethylated gene of interest using at least one primer pair, at least oneprimer of which is designed to bind only to the sequence of methylatedDNA following treatment with the reagent, wherein at least one primer inthe primer pair comprises, consists essentially of, or consists of thenucleotide sequence of any of SEQ ID NO. 14, 15, 16, 17, 18 or
 19. 15.Method of diagnosing cancer or predisposition to cancer comprisingdetecting the methylation status of the MAGE-A3 gene in a sample byusing an oligonucleotide, primer or probe as defined in claim 1, whereinthe presence of unmethylated MAGE-A3 in the sample is indicative forcancer or predisposition to cancer.
 16. Method for identifying and/orselecting a patient suitable for treatment with a MAGE-A3immunotherapeutic comprising detecting the methylation status of theMAGE-A3 gene in a sample of the patient by using an oligonucleotide,primer or probe as defined in claim 1, wherein if the MAGE-A3 gene isunmethylated the subject is identified and/or selected for treatmentwith the MAGE-A3 immunotherapeutic.
 17. Method for predicting thelikelihood of successful treatment of cancer comprising detecting themethylation status of the MAGE-A3 gene in a sample of the patient byusing an oligonucleotide, primer or probe as defined in claim 1, whereinif the gene is unmethylated the likelihood of successful treatment witha MAGE-A3 immunotherapeutic is higher than if the gene is methylated.18. Method of selecting a suitable treatment regimen for cancercomprising detecting the methylation status of the MAGE-A3 gene in asample of the patient by using an oligonucleotide, primer or probe asdefined in claim 1, wherein if the gene is unmethylated, animmunotherapeutic is selected for treatment.
 19. Method of treatingcancer in a subject comprising administration of an immunotherapeutic,wherein the subject has been selected for treatment on the basis ofmeasuring the methylation status of a MAGE-A3 gene by using anoligonucleotide, primer or probe as defined in claim
 1. 20. Method oftreating a patient comprising: measuring the methylation status of aMAGE-A3 gene by using an oligonucleotide, primer or probe as defined inclaim 1, and then administering to the patient a composition comprisingMAGE-A3.
 21. Method of treating a patient susceptible to recurrence of aMAGE-A3 expressing tumour, the patient having been treated to removetumour tissue, the method comprising: measuring the methylation statusof a MAGE-A3 gene in the tumour tissue by using an oligonucleotide,primer or probe as defined in claim 1, and then administering to thepatient a composition comprising MAGE-A3. 22.-42. (canceled)